Sensitive and rapid determination of antimicrobial susceptibility

ABSTRACT

The present invention relates to moving microorganisms to a surface, where they are grown in the presence and absence of antimicrobials, and by monitoring the growth of the microorganisms over time in the two conditions, their susceptibility to the antimicrobials can be determined. The microorganisms can be moved to the surface through electrophoresis, centrifugation or filtration. When the movement involves electrophoresis, the presence of oxidizing and reducing reagents lowers the voltage at which electrophoretic force can be generated and allows a broader range of means by which the target can be detected. Monitoring can comprise optical detection, and most conveniently includes the detection of individual microorganisms. The microorganisms can be stained in order to give information about their response to antimicrobials.

CROSS REFERENCE TO RELATED PATENT APPLICATIONS

This application is a Divisional of U.S. application Ser. No.13/763,446, filed Feb. 8, 2013, which is a Continuation of U.S.application Ser. No. 12/702,210, filed Feb. 8, 2010, now U.S. Pat. No.8,460,887, which application is a Divisional of U.S. application Ser.No. 10/888,828, filed Jul. 8, 2004, now U.S. Pat. No. 7,687,239, whichapplication claims the benefit of U.S. Provisional Patent ApplicationNo. 60/486,605, filed Jul. 12, 2003, and U.S. Provisional PatentApplication No. 60/571,479, filed May 13, 2004, all of which areincorporated by reference herein in their entirety.

TECHNICAL FIELD

The present invention relates to the rapid and sensitive detection ofthe susceptibility of microorganisms to antimicrobial drugs.

BACKGROUND

Conventional biodetection utilizes immobilized probes to detect targetsin solution. Such systems often include DNA probes to detect DNA and RNAtargets, antibody probes to detect proteinaceous, carbohydrate, andsmall organic molecule targets, and aptamer probes to detect nucleicacid, proteinaceous, carbohydrate, and small organic molecule targets.These systems can include conventional ELISA (an enzyme-linkedimmunosorbent assay) that can take place in a macrowell format (e.g. amicrotiter well), as well as microarray formats in which the immobilizedprobes can be constructed or “printed” in spots less than a hundredmicrons in diameter. Such methods are extensively practiced today inclinical and research applications (see, for example, U.S. Pat. No.5,405,783 to Pirrung, et al., U.S. Pat. No. 6,054,270 to Southern, U.S.Pat. No. 6,101,946 to Martinsky, and Weeraratna et al. “Gene ExpressionProfiling From Microarrays to Medicine”, J. Clin. Immunol. 24:213(2004), the “Packard Biochip Arrayer” from Perkin Elmer, Wellesley,Mass.).

In all of these methods, there is a binding reaction between the probeand the target, and this binding reaction is generally governed by thereaction kinetics of multiple reactant (generally bi-molecular) systems.Because the probes are immobilized, the rate of reaction is primarilydetermined by the concentration of the target in solution.

In many of the systems, the rate of the reaction is important. Forexample, in certain nucleic acid hybridizations, the reaction canrequire over 48 hours to complete, which can increase the cost of theanalysis, or reduce the number of analyses that can take place.Furthermore, if not all of the hybridizations react to completion, thenthe quantitation of the analyses can be incorrect, mixing as it wouldthe results from hybridizations at different levels of completion.

In an important application, the medical outcomes of human infections(e.g. ventilator acquired pneumonia, infectious meningitis, bacteremia,and the like) can be significantly affected by the length of timerequired to perform analysis of the amount and the identity of bacteriaand the susceptibility of the bacteria to various antibiotics.Conventionally, the time for analysis can be 24 to 48 hours or more,during which time the condition of the patient can deteriorate as thebacteria multiply (see, for example, U.S. Pat. No. 4,778,758 to Ericssonet al., U.S. Pat. No. 3,935,073 to Waters, U.S. Pat. No. 6,043,048 toJohnston et al., and U.S. Pat. No. 4,259,442 to Gayral). Contemporarymicrobial analysis starts with growth of bacteria from a clinicalspecimen, such as sputum, blood, and the like, to high concentration inculture medium, typically on the order of 100 million organisms permilliliter. Clinical specimens may contain only a few individualorganisms (e.g. in testing blood for bacteremia), and diagnosticthresholds even for high-concentration specimens are typically severalthousand-fold lower than quantitative culturing detection limits.

After achieving initial bulk growth up to an adequate workingconcentration, the operator then performs one or more biochemical testsor growth on selective media that incorporate selective biochemicalreagents. Thus the standardized current procedures require at least twosequential growth cycles, each typically requiring many hours tocomplete.

Additionally, drug susceptibility testing requires determination offailure to grow in selective media. Proof of the absence of growthrequires additional time in culture over that which might be required ofa direct indicator of cell death. It is well recognized in the medicalcommunity that such methods, attempting to prove the absence of growth,in certain circumstances produce results that do not correlateadequately with the actual results of treatment.

As a result of these and other serious deficiencies, contemporarypractice fails to provide the attending physician with specificdiagnostic information that the physician needs in order to select aneffective drug to treat the infection within the desired time window.For example, in ventilator-associated pneumonia, clinical research hasdemonstrated that the odds ratio for increased morbidity and mortalityafter 24 hours of ineffective treatment remains at 7:1 despite a changeto effective treatment. That is, unless the physician initiateseffective treatment, i.e. anti-microbial drugs of a type andconcentration adequate to quickly kill the infectious organisms, withinsubstantially less than 24 hours from symptom onset, a change fromineffective to effective therapy will not significantly improve outcomesfor approximately 87% of patients so treated.

Physicians are well aware of the risk of delay, and so prescribetreatment typically using a combination of broad-spectrum drugs selectedempirically, based on a particular hospital or community history ofmicrobial drug resistance or susceptibility. Clinical research hasdemonstrated that such empiric drug treatment is ineffective inapproximately 25% to 50% of cases. Additionally, exposure of a patientto inadequate therapy not only increases the individual patient's costsand medical risks, but also increases the likelihood of fostering theemergence of resistant organisms. The latter problem increases themedical risk not only for the individual patient, but for all otherindividuals in the hospital and community who may later become infectedwith resistant organisms.

It is well recognized in the clinical research literature that priorexposure of a patient to ineffective antibiotics constitutes asignificant risk factor in the later emergence of resistant organisms inthat patient. For these and other reasons, it is desirable within themedical community to devise diagnostic methods that do not suffer thedeficiencies of delay and inaccuracy that characterize currentpractices.

In theory, alternatives to microbial growth culturing include directmicrobial analytical methods such as immunoassays of various kinds.Antibodies against various microbes are commercially available or may bereadily developed. In fact, many different types of immunoassay are nowroutinely used in certain aspects of diagnosis for microbial infection.

However, none yet exist for routine bacterial identification,quantitation, and drug susceptibility testing for many seriousinfectious diseases.

Similarly, the rapid detection of various microbes such as bacteria,viruses, molds, and the like are also desirable for testingcontamination in food and water, and in detecting the presence ofpotential biological warfare agents. In the food industry, many productsare commercially available for detecting microbial contaminants. Incertain circumstances, some of these provide results in approximately 24hours for a limited set of particular organisms. However, all commercialproducts still require sample enrichment by means of bacterial culturingbefore applying the tests.

In the research literature concerning defense for biological warfare,many rapid detection devices have also been described, including somethat provide results in one hour or even less. Furthermore, some suchdevices do not require growth cultures before being used.

However, the sensitivity of devices so far described in the literaturefor food testing or bio-defense falls far short of the requirements formedical diagnostics. Furthermore, these non-diagnostic applications donot require drug susceptibility testing and so the aforesaid devices donot provide it nor apparently do they lend themselves to adaptation forsuch a purpose.

A key limitation with these devices and with laboratory methods such asELISA is their dependency on the target analyte concentration. They relyon passive diffusion of target to an immuno-capture or other detectionsurface. The rate of occurrence of intimate probe-to-target proximityevents, and hence the detection reaction rate, depends on analyteconcentration in the sample solution or suspension.

In order to increase sensitivity with these devices, it is necessary tosubstantially increase analyte concentration. Researchers have describedseveral stratagems to increase target analyte concentration and alsospeed the response time for analysis of various bio-molecular andmicrobial targets. For example, the electrophoresis of target to theprobe has been described before by Nanogen, Inc. of San Diego, Calif.(e.g. U.S. Pat. No. 5,849,486 to Heller, U.S. Pat. No. 6,017,696 toHeller, U.S. Pat. No. 6,051,380 to Sosnowski et al., U.S. Pat. No.6,099,803 to Ackley et al., U.S. Pat. No. 6,245,508 to Heller et al.,and U.S. Pat. No. 6,379,897 to Weidenhammer et al.). These systems andmethods describe an addressable array of electrodes to which individualprobes are attached at each individual electrode, and then which aresequentially and very rapidly reacted with probes. The reported increasein speed of reaction between the target and probes is hundreds orthousands fold. These systems, however, suffer from a number oflimitations, including the need to sequentially immobilize probes on theaddressable electrodes, the need to perform sequential reactions, andlimitations on the detection methods that can be employed due to thehigher voltages that are required for electrophoresis, precluding theuse of transparent electrodes (e.g. through the use of indium tinoxide), that cannot operate at the voltages used by the Nanogen system.Furthermore, the higher voltages at which the Nanogen system operategenerate oxidation products that are potentially harmful to the probesor targets, and which therefore requires the use of complex passivationsurfaces to protect the probes and targets. Systems that could make useof high-speed microarray printing, which did not require complexpassivation surfaces, and which did not require the electronic and othercontrol necessary for addressable electrodes would greatly reduce theexpense and complexity of such systems.

With regards to the use of immobilized probes for the detection ofbacteria or other microorganisms, it is also of use to determine theantimicrobial activity of different therapeutic agents, such asantibiotics. There has been a profusion of systems that use nucleic acidor antibody probes to determine the identity of bacteria in a sample(e.g. U.S. Pat. No. 5,656,432 to Clayerys et al. and U.S. Pat. No.6,403,367 to Cheng et al.). It is difficult with these systems todetermine susceptibility to antimicrobial agents, given the difficultyof finding nucleic acid or antibody markers that reliably correlate withantimicrobial resistance or behavior.

It is to the solution of these and other problems that the presentinvention is directed.

SUMMARY OF THE INVENTION

In light of the deficiencies of existing biodetection systems andmethods, it is an objective to perform detection of biological moleculesrapidly.

It is additionally an object of this invention to minimize nonspecificbinding that reduces the sensitivity of biodetection.

It is also an object of this invention to be able to distinguishspecific from nonspecific binding of a target to a surface.

It is another object of this invention to be able to identifymicroorganisms and to determine their susceptibility to anti-organismagents.

It is further an object of this invention to capture probes rapidly ontoa surface in order to permit their detection.

Additional objects, advantages and novel features of this inventionshall be set forth in part in the description that follows, and in partwill become apparent to those skilled in the art upon examination of thefollowing specification or may be learned by the practice of theinvention. The objects and advantages of the invention may be realizedand attained by means of the instrumentalities, combinations, andmethods particularly pointed out in the appended claims.

In summary, the invention comprises processes and components that can beused singly or in combination for beneficial effect. The resultingmethods and devices can be used in the biodetection of a variety ofdifferent analytes within a sample, including nucleic acids, proteins,starches, lipids, and organisms and cells. In the most general forms,these entities are captured onto a substrate, where they are detected.

One aspect of the present invention involves the detection ofmicroorganisms on a fixed substrate at more than one time. Changes inthe conditions of the microorganisms at the different times can indicatetheir response to agents to which the microorganisms are exposed. Theconditions of the bacteria can include their appearance with variousstains, such as vital and mortal stains, or the appearance of growth inthe microorganism, either through its size, ability to accept additionalstaining agent, or the occurrence of nearby “daughter” microorganismsthat indicate the doubling of the microorganisms. More generally, thecondition can include the identity of the microorganism, as might beindicated by a serological stain. The agents can include a variety ofdifferent antibiotics, which can be provided to the microorganisms at anumber of different concentrations in order to determine properties ofthe bacteria such as the minimum inhibitory concentration or the minimumbacteriocidal concentration. The microorganisms can be challenged notonly with constant concentrations of the agent, but the agent can alsobe exposed to a varying concentration that can mimic thepharmacokinetics of the agent.

It should be noted that looking at the growth and behavior of individualmicroorganisms has great beneficial effect, given that most currentmeans of monitoring microorganisms requires a large number ofmicroorganisms, and it can take an extended period to grow to sufficientnumbers of microorganisms. By monitoring individual microorganisms, itis not even required for all of the individual microorganisms to showthe effect, but only for a sufficient fraction so that the effect isdemonstrated over a statistical background. This can allow for a veryrapid test.

Another aspect of the present invention is the movement of themicroorganisms or other analytes to a substrate where they can becaptured. This movement can comprise a number of different forces,including electrophoresis, dielectrophoresis, centrifugation, magneticfield capture, filtration, gravity or diffusion. In many instances, thenaturally occurring forces of gravity and diffusion are not strongenough for the movement to occur in a practical time for the test, andtherefore the application of other artificial forces are necessary. Theforces can act either directly on the analyte, or the analyte can bebound to a tag that responds to the application of the artificial force.The tag can comprise an electrostatic tag, which can include apolyelectrolyte, which then moves in an electrophoretic ordielectrophoretic field. The tag can also comprise a paramagneticparticle that responds to a magnetic field.

A further aspect of the present invention is to use a movement of theanalyte with a component parallel to the surface where the analyte iscaptured either at the same time as or interspersed with the movement ofthe analyte towards the surface. This allows the analyte to becomedistributed along the surface, and can further allow for a largerfraction of the analyte to bind where there are multiple regions ofpotential binding. If these regions have different specificity fordifferent species of analyte within the sample, then this allows theanalyte to be moved from region to region until it contacts the regionwith the matching specificity. The movement parallel to the surface cancomprise electrophoresis, filtration, or bulk flow (which can beinstituted, for example, by pumps, electroosmosis, or other means).

Another aspect of the present invention is to tag the analyte with anindicator that confers detectability on the analyte. The indicator cancomprise a light scattering particle, an enzyme-containing particle, afluorophore, an upconverting phosphor, a quantum dot, or anelectrochemical agent. It can also be very useful to have a tag thatconfers both detectability as well as movement with an artificial force(as described above).

A yet further aspect of the present invention is to remove the analytethat is nonspecifically bound to the surface. This washing can utilizethe same forces that move the analyte towards the surface, but nowapplied in another direction. Such forces can include electrophoresis,dielectrophoresis, and magnetic forces. The forces can also comprisephysical and chemical conditions such as pH, ionic strength, and bulkflow (laminar or otherwise).

It is also an aspect of the present invention that there be frequentmonitoring of the analyte on the surface. For example, it is preferablefor there to be a number of different stringencies of removal of thenonspecifically bound material, so that specifically-bound material canbe distinguished both from nonspecifically-bound material that isless-forcefully bound as well as from material that is more-forcefullybound. The frequent monitoring can then identify specifically-boundmaterial by looking at the stringency at which different material isremoved from the surface.

An aspect of the present invention is monitoring in real-time usingoptical methods, which can not only identify the presence of an analyteon the surface, but also to store the location of individual analytes onthe surface so that its presence can be monitored over time. The opticaldetection can comprise imaging detectors, such as a camera, but can alsocomprise a scanning laser with a light detector, that can be a photomultiplier tube. The detector can detect either the analyte itself, oras described above, an indicator that is bound to the analyte. Thedetector can comprise a brightfield, darkfield, phase, fluorescent, orother emitted light detector. Alternatively, the detector can comprise asurface plasmon resonance detector, wherein the surface comprises gold.

An additional aspect of the present invention relates to the use ofindium tin oxide and other transparent conductive coatings whichfacilitate the use of optical detection. In these cases, it is necessarythat the voltages that are used not exceed on the order of 2 Volts,which potential does not support electrophoresis and dielectrophoresiswith many conventional buffers. It can be convenient to use redoxreagents in order to support electrophoresis and dielectrophoresis.These redox reagents can be in pairs, in which the oxidation of thereducing agent gives rise to the oxidizing agent, and the reduction ofthe oxidizing agent gives rise to the reducing agent. Other arrangementsare also possible, for example in which the oxidation product of thereducing agent oxidizes the reduction product of the oxidizing agent. Itis also convenient for these reagents to be neutrally charged, so thationic species do not interfere with the electrophoresis anddielectrophoresis.

It is yet an additional aspect of the present invention for thesolutions in which electrophoresis and dielectrophoresis occur to havelow ionic strength, so that the electrolytes do not reduce theeffectiveness of the electrophoresis. In these cases, it is convenientfor the solutions to comprise zwitterionic species both for buffering,for stabilizing the interactions between molecular species, and forproviding a growth conducive environment for microorganisms.

Another aspect of the present invention is for the illumination tocomprise evanescent wave illumination, since this detects only thatanalyte that is juxtaposed with the surface, and thus analyte orindicators that are not bound can remain in the solution above thesurface. The evanescent wave illumination can be coupled into thesubstrate beneath the surface using gratings, end-couplings, and prisms.While the evanescent wave illumination can bounce multiple times withinthe substrate, it is also convenient for the evanescent waveillumination to have a single bounce against the surface, which isconveniently performed with prisms which can be either detachable orpermanently attached or formed with the substrate. If detachable, theinterface between the prism and the substrate can be a transparent,elastic material.

A yet further aspect of the present invention is the use of samplepresentation, which can comprise concentration of the analyte from alarge sample volume, as well as removal of contaminants. This samplepreparation can comprise centrifugation, ion exchange beads or columns,filtration, stacking electrophoresis, or forms of biochemicalseparation.

As described above, numerous embodiments of the present invention can beassembled from these and other aspects of the present invention. Forexample, one preferred embodiment resulting from the combination ofaspects of the present invention relates to a system for thequantitation of microorganisms of a first type in a solution. Thissystem comprises a chamber comprising a first electrode and a secondelectrode on opposing walls of the chamber, an input port, an outputport, and a fluid transport means for transporting solution into thechamber through the input port and out of the chamber through the outputport. The system further comprises a first affinity component affixed tothe first electrode, to which microorganisms of the first type adhere,an electrical controller that controls the potential between the firstelectrode and the second electrode, an automated detector that candetect the quantity of microorganisms of the first type adhered to thefirst affinity component, and an information controller that stores thequantity of microorganisms of the first type as determined by thedetector. In the system, the solution is introduced into the chamberthrough the input port, a potential is applied by the controller betweenthe first and the second electrodes sufficient to cause electrophoresisto occur between the electrodes, causing movement of microorganisms ofthe first type towards the first electrode to occur, such that when themicroorganisms are proximate to the first affinity component, they bindto the first affinity component and their quantity is determined by thedetector and stored in the information controller.

The microorganisms may comprise bacteria selected from a set of generasuch as Pseudomonas, Stenotrophomonas, Acinetobacter, Enterobacter,Escherichia, Klebsiella, Proteus, Serratia, Haemophilus, Streptococcus,Staphylococcus, Enterococcus, Mycobacterium, Neisseria, and other humanpathogens encountered in medical practice. Similarly, microorganisms maycomprise fungi selected from a set of genera such as Candida,Aspergillus, and other human pathogens encountered in medical practice.Still other microorganisms may comprise human pathogenic virusesencountered in medical practice.

The oxidizing agent may comprise benzoquinone, a dithiol, a ketone, aferrocinium, a ferricyanide, dihydroascorbate, oxidized glutathione,oxidized methyl viologen, or a halogen. The reducing agent may comprisedithiothreitol, dithioerythritol, a dithioalkane, a dithioalkene, athioalkane, a thioalkene, a thiol, a hydroquinone, an alcohol, aferrocene, a ferrocyanide, ascorbate, glutathione, methyl viologen, or ahalide. Also, the reduced product of the oxidizing reagent may comprisethe reducing agent.

The conductivity of the solution may be less than 100 microSiemens/cm orthe conductivity of the solution may be less than 10 microSiemens/cm.The solution may comprise a zwitterionic buffer.

A concentrator may concentrate the microorganisms from a sample. Theconcentrator may comprise a centrifuge. The concentrator may compriseion exchange particles.

The sample may have a higher conductivity than the solution.

The automated detector may comprise an optical detector. The opticaldetector may utilize optical detection methods including lightscattering imaging, brightfield imaging, darkfield imaging, surfaceplasmon resonance, phase imaging, fluorescence imaging, upconvertingphosphor imaging, quantum dot imaging, and chemiluminescence imaging.

An electrode selected from the set comprising the first electrode andsecond electrode may be optically transparent.

The target may be illuminated by a laser.

The detector additionally may determine the position of eachmicroorganism adhered to the affinity component, wherein the locationsof the microorganisms may be stored in the information controller alongwith the quantity of the microorganism at that location.

The detector may detect total amount of microorganisms of the first typethrough averaging of signal of a portion of the surface comprisingsubstantially all of the microorganisms of the first type affixed to thefirst electrode.

The first electrode may be comprised of gold, and the detector mayutilize surface plasmon resonance.

The detector may comprise a camera.

The field of view corresponding to each pixel may comprise a long axisthat is less than 2 microns, or may be less than 0.5 microns.

The solution may be in bulk movement during electrophoresis.

Two periods of electrophoresis may be interspersed with a period inwhich the solution is in bulk movement.

The solution additionally may include microorganisms of a second type,wherein the detector can distinguish microorganisms of the first typefrom microorganisms of the second type.

A first tag may be comprised of a first binding agent linked to a firstindicator that is detectable by the detector and a second tag may becomprised of a second binding agent linked to a second indicator that isdetectable by the detector and wherein the first indicator and thesecond indicator are distinguishable by the detector, wherein the firstbinding agent binds to microorganisms of the first type, and the secondbinding agent binds to microorganisms of the second type, wherein thefirst tag and the second tag are reacted with microorganisms of thefirst type and microorganisms of the second type bound to the affinitycomponent, and the detector substantially simultaneously detects thequantity of the microorganisms on the basis of the tags that are boundto the microorganisms.

A first tag may be comprised of a first binding agent linked to anindicator that is detectable by the detector and a second tag iscomprised of a second binding agent linked to the indicator, wherein thefirst binding agent binds to microorganisms of the first type, and thesecond binding agent binds to microorganisms of the second type, whereinthe first tag is reacted with microorganisms of the first type bound tothe affinity component and the detector detects the quantity andlocation of the microorganisms of the first type on the basis of thetags that are bound to the microorganisms, and subsequently, the secondtag is reacted with microorganisms of the second type bound to theaffinity component and the detector detects the quantity and location ofthe microorganisms of the second type on the basis of the tags that arebound to microorganisms that are in locations that were not previouslydetected by the detector.

A tag may be comprised of a binding agent linked to an indicator,wherein the binding agent may comprise an antibody that binds tomicroorganisms of the first type.

The detector may distinguish microorganisms of the first type frommicroorganisms of the second type on the basis of differingelectrophoretic properties of the microorganisms.

The first affinity component may comprise a polyelectrolyte. Thepolyelectrolyte may comprise a polycationic polymer. The polycationicpolymer may comprise amine moieties. The polymer may comprisepolyethyleimine or polylysine.

The solution additionally may include microorganisms of a second type,wherein microorganisms of the second type do not bind to the firstaffinity component. The affinity component may comprise an antibody oran aptamer.

A second affinity component may be bound to the first electrode, towhich microorganisms of the second type adhere, wherein the detector candetect the quantity of microorganisms of the second type adhered to thesecond affinity component, wherein the system can distinguishmicroorganisms of the first type from microorganisms of the second typeby whether the microorganisms adhere to the first affinity component orthe second affinity component.

The affinity component additionally may comprise a polymer that hasintrinsically low affinity for microorganisms, wherein the polymer maycomprise polyethylene glycol or polyacrylamide.

The system may additionally comprise a third electrode, co-planar withthe first electrode, to which a second affinity component may bind andto which microorganisms of the second type may adhere, wherein thepotential on the first electrode and the third electrode may beindependently controlled by the electrical controller.

The detector may detect whether microorganisms of the first type arelive or dead. The microorganisms may be stained prior to being detectedby the detector with a mortal stain or the microorganisms may be stainedprior to being detected by the detector with a vital stain. Subsequentto the microorganisms of the first type adhering to the first affinitycomponent, the microorganisms may be placed in conditions conducive togrowth. These conditions may comprise temperatures between 34 and 40degrees C.

The solution may be removed from the chamber via the output port and maybe replaced by growth medium through the input port. Also, the growthmedium may have a conductivity of less than 1 milliSiemens/cm, and theelectrical controller may maintain a potential of greater than 100 mVbetween the first electrode and the second electrode.

Microorganisms of the first type may be detected by the detector at aninitial time, and may also be detected at a second time after themicroorganisms are allowed growth time sufficient for at least 10% ofthe microorganisms to double, wherein differences in the detectedmicroorganisms of the first type at the second time may provide evidenceof the viability of the microorganisms of the first type in the growthconditions. Also, an anti-microorganisms agent may be added to thegrowth medium during the growth time. The detector may detect ifmicroorganisms of the first type are live or dead in response to theanti-microorganism agent, wherein prior to detection by the detector themicroorganisms are stained with a stain selected from the set consistingof mortal stain and vital stain. The anti-microorganism agent maycomprise individual agents or combinations of agents selected fromantibiotic families such as cephalosporins, penicillins, carbapenems,monobactams, other novel beta-lactam antibiotics, beta-lactamaseinhibitors, fluoroquinolones, macrolides, ketolides, glycopeptides,aminoglycosides, fluoroquinolones, rifampin, and other families,including novel agents, used as antibiotics in clinical practice or inresearch. Also, the concentration of the anti-microorganism agent may bechanged over time to reflect the pharmacokinetics of theanti-microorganism agent in animal tissue.

The microorganisms may be reacted with a surplus of microorganismsurface binding reactants at a first time period, after which thereactants are subsequently removed, and wherein at a second time periodthe microorganisms may be reacted with a surplus of microorganismsurface binding molecules modified by an indicator so as to bedetectable by the detector, wherein the detection of the indicator bythe detector indicates the growth of the microorganisms.

The solution additionally may comprise a contaminant that binds to thefirst affinity component along with the microorganisms of the firsttype, wherein a condition is applied to the first affinity componentwhich releases the contaminant without releasing the microorganism,whereas the contaminant is removed by application of the condition. Thecondition may comprise temperature, magnetic field strength,electrophoretic force, dielectrophoretic force, shear fluid flow, ionicstrength, pH, non-ionic surfactant concentration, ionic surfactantconcentration, or competitor concentration. The solution additionallymay comprise a contaminant which binds to the first affinity componentalong with the microorganisms of the first type, wherein a condition isapplied to the first affinity component which releases themicroorganisms without releasing the contaminant, whereas themicroorganisms may be subsequently bound to a second affinity componentaffixed to the first electrode. The condition may comprise temperature,magnetic field strength, electrophoretic force, dielectrophoretic force,shear fluid flow, ionic strength, pH, non-ionic surfactantconcentration, ionic surfactant concentration, or competitorconcentration.

The microorganisms of the first type may be concentrated in the solutionprior to being bound to the first affinity component, wherein themicroorganisms are present in a first salt buffer of relatively lowionic strength, and the first salt buffer is proximal to a second saltbuffer of relatively higher ionic strength and the first salt buffer andthe second salt buffer adjoin at an interface, and wherein a firstconcentration electrode is located proximal to the interface and asecond concentration electrode is located distal to the interface,wherein the placement of a potential between the first concentrationelectrode and the second concentration electrode causes themicroorganisms to migrate through the first salt buffer byelectrophoresis and their migration is reduced more than X fold uponmeeting the interface. The second concentration electrode may comprisethe first electrode. Also, the interface may be located substantially atthe input port. The ratio of conductivity between the first salt bufferand the second salt buffer may be less than 1:50.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of a biodetection system that utilizes aprobe having affinity for a target.

FIG. 2A, a schematic diagram of a biodetection system taking place inwhich different probes 116 are placed in an array of locations on asubstrate.

FIG. 2B is a side-view through the array of FIG. 2A.

FIG. 3 is a perspective diagram of an electrophoretically-enhancedincubation system.

FIG. 4A is a perspective diagram of a biodetection system wherein asingle probe electrode underlies multiple probe locations which areplaced into an array.

FIG. 4B is a perspective diagram of a biodetection system wherein theelectrodes do not underlie the probe locations.

FIG. 5 is a diagram of electric field strengths from a first electrode,a second electrode, and a set of partial reference electrodes.

FIG. 6 is a schematic diagram of an electrophoretic tag in a sandwichconfiguration.

FIGS. 7A through F are schematic diagrams of electrophoretic tags,showing differing arrangements of components to provide similarfunctionality.

FIG. 8 is a schematic block flow diagram of the steps of the presentinvention.

FIG. 9 is a graph of the amounts of material bound versus the bindingforce.

FIG. 10 is a schematic block flow diagram of a system involvingelectrodes not underlying probe locations.

FIG. 11A is a schematic block flow diagram of the operation of a cellinvolving electrodes underlying probe locations, and can be bestunderstood in relation to FIG. 4A.

FIG. 11B is a schematic flow diagram of the operation of a cellinvolving electrodes under the probe locations using a tagged target.

FIG. 12A is a schematic diagram of three electrodes arranged on twoperpendicular axes within a reaction cell.

FIG. 12B is a graph of the potential difference between the electrodesE2 and E4 as they vary with time, with electrode E4 biased positively toE2.

FIG. 12C is a graph of the potential difference between the electrodesE2 and the four as they vary with time, arranged alternatively to thatin FIG. 12B.

FIG. 12D is a graph of potential differences between spatially displacedelectrodes, such the electric field changes not only magnitude but alsoin direction.

FIG. 13A is a schematic diagram with three electrodes displaced in twodimensions over a single electrode 200.

FIG. 13B is a graph of potential differences between the electrodes ofFIG. 13A.

FIG. 14A is a schematic diagram of a closed system for electrophoresis.

FIG. 14B is a schematic diagram of an open system for electrophoresis.

FIG. 15 is a top-view schematic of a region in which cell ofinhomogeneity have formed.

FIG. 16A is a schematic block diagram of a reaction involving bothvertical forces and horizontal forces so as to accelerate the reactionof a tagged target with the probe 116.

FIG. 16B is a graph of the electrical potential causing movement of thetagged target vertically, in time relation to the horizontal forcescausing mixing of the tagged target.

FIG. 17A is a schematic block diagram of the means of controlling thehorizontal and vertical forces.

FIG. 17B is a schematic block flow diagram of the operation of thesystem of FIG. 17A.

FIG. 18A is a perspective diagram of a mechanical stirring system thatcan be used within a microtiter plate well.

FIG. 18B is a top-view diagram of the probe electrode.

FIG. 19A is a perspective diagram of a microtiter plate with a set ofelectrodes 570 and shafts 552.

FIG. 19B is a perspective view of a top plate comprising access ports.

FIG. 20A is a top view of the arrangement of well electrodes on a bottomplate 592.

FIG. 20B is a top view of the arrangement of electrically-connected wellelectrodes on a bottom plate.

FIG. 21 is a schematic drawing of a cross-section of a detection systemcomprising a detection sandwich on a substrate.

FIG. 22 is a schematic block flow diagram of discrimination usingelectrophoretic force.

FIG. 23A is a cross-sectional schematic of an embodiment of the presentinvention in which a prism on the top surface is used to introduce lightinto the slide waveguide.

FIG. 23B is a cross-sectional schematic of a prism on the top surface ofa slide, in which light is internally reflected within the prism priorto introduction of the light into the slide waveguide.

FIG. 24A is a cross-section schematic of the prism arrangement of FIG.3, extended so that the disposition of the distal parallel ray paths canbe seen.

FIG. 24B is the cross-sectional schematic of FIG. 4A, modified by theuse of convergent illumination instead of collimated illumination.

FIG. 24C is a schematic cross-sectional diagram of a slide illuminatorin which the slide is non-uniformly illuminated.

FIG. 25A is a schematic cross-section of an end-illuminated thin-filmwaveguide integrated with a slide.

FIG. 25B is a schematic top view of the coupler and the slide of FIG.5A.

FIG. 25C is a schematic cross-section of a thin film waveguide whereinlight is coupled to the waveguide via a grating.

FIG. 25D is a schematic cross-section of a thin film waveguide whereinlight is coupled to the waveguide via a high-index material prism.

FIG. 26A is a schematic cross-section of evanescent illumination of aregion without use of a waveguide.

FIG. 26B is a schematic cross-section of evanescent illuminationaccording to FIG. 6A, in which the prism has a window through which thedetector detects reporters on the top surface of the slide.

FIG. 27A is a schematic cross-section of light coupling with a prismusing a flexible coupler.

FIG. 27B is a side-view schematic of a prism with a curved face coupler.

FIG. 28A is a graph of the washing potential as a function of time for asimple step washing function.

FIG. 28B is a graph of the washing potential as a function of time for aramped washing function.

FIGS. 29A-B are schematic diagrams of a tagged target comprising asingle-stranded DNA target binding to a complementary DNA probe, whichis bound to the substrate at one or more points of attachment.

FIG. 30A is a schematic side-view diagram of two reference electrodesrelative to the probe electrode.

FIG. 30B is a graph of the potential of electrode relative to the tworeference electrodes as shown in FIG. 30A for two steps in the washingstringency.

FIG. 31 is a block flow diagram of the process for determining theidentity, number and antibiotic sensitivity of bacteria in a sample.

FIG. 32A is a top schematic diagram of a bacterial detection cell.

FIG. 32B is a side-view schematic diagram of the bacterial detectioncell of FIG. 32A through the cross-section X.

FIG. 32C is a side-view schematic diagram of the bacterial detectioncell of FIG. 32B with the use of addressable electrodes.

FIGS. 33A-F are side schematic views of the transport and capture ofbacteria using the chamber of FIGS. 32A-B.

FIGS. 34A-D are side-view schematic diagrams of electrophoretictransport to the detection surfaces.

FIGS. 35A-D are side-view schematic diagrams of a chamber in whichcontaminating material is distinguished on the basis of its behaviorunder electrophoretic fields.

FIGS. 36A-E are side-view schematic diagrams of detection of multiplebacteria on a nonspecific surface.

FIGS. 37A-D are schematic diagrams of detecting growth in an organism.

FIGS. 38A-B are graphs of the response of bacteria to a changingconcentration of an anti-organism agent.

FIG. 39A is a schematic view of a centrifuge tube modified for theconcentration of bacteria onto a capture surface.

FIG. 39B is a cross-sectional view of the centrifuge tube of FIG. 39A.

FIG. 39C is a cross-sectional side-view of a detector using the capturepiece of FIGS. 39A-B.

FIGS. 40A-B are a cross-sectional top-view and side-view of a detectionsystem that uses a porous capture filter.

FIGS. 41A-B are schematic cross-sections of a detection system usingmultiple forces to effect separation of the bacterial sample.

FIG. 42 is a block diagram of a biodetection by the present invention.

BEST MODE FOR CARRYING OUT THE INVENTION Biodetection Background

FIG. 1 is a schematic diagram of a biodetection system 100 that utilizesa probe 116 having affinity for a target 114. The probe 116 is affixedto a solid substrate 120 by a probe linker 118. The probe linker 118will generally comprise a coating that further serves to reduce theadventitious binding of target molecules to the substrate 120. Thetarget 114 is connected via a target linker 112 to a tag 110, which canbe detected by a detector, not shown.

The target 114 can comprise a variety of biomolecules, including nucleicacids, proteins, starches, lipids, hormones, and more. Furthermore, thetarget 114 can comprise, as will be discussed below in greater detail,whole organisms or organelles, including bacteria, fungi, viruses,mycoplasmas, cell fractions (mitochondria, nuclei), animal or plantcells, and other organisms. In each case, the probe 116 will match thetarget 114, and itself can comprise nucleic acids (both forhybridization and as aptamers), proteins, carbohydrates, and can alsoinclude whole organisms and organelles as described above. Indeed, inmost cases, wherever one has a target-probe pair, the constituents cangenerally be switched so that the target acts as a probe, and the probeas a target, on the basis of their affinity for each other.

In operation, the target 114, which is connected to the tag 110, isintroduced into solution that is in contact with the probe 116. Becauseof the molecular affinity of the probe 116 for the target 114, thetarget 114 binds to the probe 116. Because the tag 110 is attached tothe target 114, the presence of the tag 110 in proximity to the surfaceof the substrate 120 indicates the presence of the target 114. Bydetermining the amount of the tag 110, the amount of the target 114 canbe estimated.

Alternatively, the tag 110, instead of being bound directly to thetarget 114, can be attached via a linker to a second molecule withaffinity for the target 114. After incubation with the probe 116 and thetarget 114, a “sandwich” is formed in which the target 114 associateswith both the probe 116 and the tag 110.

One of the difficulties of the systems according to FIG. 1 is the timethat it takes for the incubation of the target 114 and the probe 116 tocome to dynamic completion. Consider, for example, a common microplatelaboratory format in which different probes are placed in a grid ofwells arranged in an eight column by twelve row well format (as will bedescribed in greater detail below). The plate well layout is defined byindustry standards and the wells are typically on the order of 9 mm indiameter. The binding of the target to the probe requires the twospecies to be in close proximity measured on a scale of Angstroms. In atypical microplate assay, diffusion and sometimes convection areutilized to increase the probability that the two species come in closeproximity to complex at the surface. This strategy generates significantsignal, at hours long incubation, with typical conventional detectionmethodologies at pg/ml concentrations of a 50 kd model protein. However,at sub or low pg/ml concentrations, the signal generation is limited bythe mass transport of analyte to surface, so that unreasonable reactiontimes measured in days are required for the assay to reach completion.

Furthermore, transport of target 114 to probe 116 is further exacerbatedin a micrometer scale array of probes (i.e. microarray format). FIG. 2Aillustrates a schematic diagram of a biodetection system in whichdifferent probes 116 are placed in an array 140 of locations 130 on asubstrate 120. Each location 130 is typically on the order of 50 micronsto 500 microns in diameter, and with an array comprising ten to tens ofthousands of locations 130, a typical side-to-side dimension for thearray 140 can be millimeters or even centimeters. The binding of thetarget 114 to the probe 116 requires that the two species be in closeproximity measured in Angstroms. Given that the passive diffusion oflarge biological macromolecules is low (e.g. measured in nanometers persecond), the lateral movement of the target 114 to the probe 116 cantake on the order of tens of hours, unless active assistance isprovided.

Even with assisted movement of the molecules laterally, the verticalscale of the incubation can frustrate the target 114 to probe 116binding. Consider FIG. 2B, a side-view through the array 140 of FIG. 2A.A cover 111 comprises the top of the incubation cell, and if the target114 is near the top of the incubation chamber (delimited by thesubstrate 120 and the cover 111), the vertical dimension is still largeby molecular standards. Consider that the smallest vertical thicknessused in conventional incubations is typically about 50 microns. Giventhat the target 114 and the probe 116 need to be within a few Angstroms,in general, in order to bind to one another, the vertical scale is10,000 times this size. In the best of cases, the target 114 would belimited in its movement to a very small volume in the vicinity of theimmobilized probe 116 to increase its apparent concentration.

A prior art embodiment of a means to overcome this problem is providedin FIG. 3, a perspective diagram of an electrophoretically-enhancedincubation system. In this case, the different probes 116 are affixeddirectly onto electrically conductive electrodes 150. These electrodes150 are independently voltage-biased relative to a reference electrode140 so as to cause a current within the incubation chamber, in whichtarget 114 molecules migrate to the electrode 150. Consider, forexample, that electrode 150 A is initially voltage biased to attract thetarget 114. Because of the immediate proximity of the target 114 and theimmobilized probe 116 at the electrode 150, the binding between the twospecies occurs very rapidly—on the order of seconds to tens of seconds.The voltage on the electrode 150 A is then made neutral or opposite toits previous bias, and the electrode 150 B is then biased. In this case,the target 114 molecules would migrate to the second electrode 150 B soas to allow the interaction of the target 114 with the probe immobilizedin the second location.

This embodiment has been extensively used by Nanogen (San Diego,Calif.), and the prior art teachings are specified in a series ofpatents, including U.S. Pat. No. 5,849,486 and U.S. Pat. No. 6,017,696.There are a number of limitations of this embodiment, however. Forexample, the area covered by the probe 116 and the respective electrode150 must be exactly coincident. In general, this means that the probes116 are immobilized sequentially using movement of the probes 116analogous to the movement of the target 114 during the incubation.Furthermore, each probe 116 electrode 150 must establish its ownelectrical connection to a power controller, which requires bothsophisticated manufacturing and power control.

Arrangement of Components

Some embodiments of the present invention comprise the application ofelectrophoretic forces on the target 114 wherein the electrodes involvedin such forces are not necessarily coincident with the locations onwhich the probe 116 is attached. The application of electrophoreticforce can be according to a number of embodiments, of which two arepresented for discussion purposes: firstly, in which the electrodes donot underlie the probe locations 116 whatsoever, and wherein theelectrophoretic forces are primarily lateral to the surface of thesubstrate 120, and secondly, in which a single electrode underlies aplurality of probe 116 locations. It should be noted that the structuralarrangement of the probe locations and the electrodes giving rise to theelectrophoretic forces will be first considered, along with variouscomponents optimized for use with the present invention, and thereafterthe operation of the various components in concert will be described. Itshould also be noted that dielectrophoresis rather than electrophoresiscan be used to move targets (or tags that are attached to targets) thatare large and electrostatically polarizable. These methods generallyrequire the use of electrodes that are shaped either in two or threedimensions so as to create electrical or electrophoretic fields that arenon-uniform. A description of the use of these dielectrophoreticelectrodes is presented in G. H. Markx and R. Pethig, DielectrophoreticSeparation of Cells: Continuous Separation. Biotechnol. Bioeng. 45,337-343 (1995) and G. H. Markx, Y. Huang, X.-F. Zhou and R. Pethig,Dielectrophoretic characterization and separation of micro-organisms,Microbiology, 140, 585-591 (1994).

Arrangement Involving a Single Electrode Underlying Multiple ProbeLocations

FIG. 4A is a perspective diagram of a biodetection cell wherein a singleprobe electrode 200 underlies multiple probe locations 170 which areplaced into an array 180. The walls of the cell are not placed in thediagram, and will generally comprise gasket material to form a watertight seal. A reference electrode 190 is physically placed preferablyabove the probe electrode 200 and of roughly similar size to the probeelectrode 200, so that the electric field between the two electrodes issubstantially uniform. However, it is also within the spirit of thepresent invention for the reference electrode 190 to have various shapesand positions that allow for similar or even lesser uniformity. Ingeneral, the electrodes are roughly parallel to one another, so that theelectrophoretic fields that are generated are roughly perpendicular tothe surface of the probe electrode 200, and give rise to even depositionof the targets onto the probe locations 170.

This arrangement of the probe electrode 200 and the probe locations 170allow for standard methods of placement of probes on the electrodesurface using contact or non-contact (e.g. pin or piezoelectric)spotters. Furthermore, the association of the target 114 with the probe116 can be performed in parallel with all of the different probelocations 170, rather than serially as performed with the prior art.

Arrangement Involving Electrodes not Underlying Probe Locations

An alternative arrangement is shown in FIG. 4B, a perspective diagram ofa biodetection cell wherein the electrodes do not underlie the probelocations 170. In this case, the probes 116 are placed in probelocations 170 arranged in an array 180. A first electrode 210 and asecond electrode 220 are lateral to the array 180, and sit underneath anarray of partial reference electrodes 195, labeled in this figure P, Q,and R. The number and type of partial reference electrodes 195 can bevaried, and the goal of the placement of the first electrode 210, thesecond electrode 220, and the partial reference electrodes 195, is tomanage the strength and topology of the electric fields by adjusting therelative voltages of the electrodes. For instance, placing the secondelectrode 220 and the partial reference electrodes 195 P, Q and R at anegative bias, and the first electrode 210 at a relatively positive biaswill cause a largely horizontal electric field across the surface of thearray 180. The need for the multiple partial reference electrodes 195 isdue to the “shorting” of the electric field that would occur with alarge, continuous electrode, making it difficult to maintain an electricfield across a larger electrode.

FIG. 5 is a diagram of electric field strengths from a first electrode210, a second electrode 220, and a set of partial reference electrodes195. The second electrode 220 and the partial electrodes 195 have anegative bias, and the first electrode 210 has a relatively positivebias. As can be seen, the vertical component of the electric field atthe location of the array 180 is relatively constant with a downwardscomponent. By adjusting the relative strengths of the voltage bias atthe different electrodes, a variety of different electric fieldtopologies can be arranged for purposes that will be described below.

Electrophoretic Tags

Most biological molecules have associated electrostatic charge, whichcan be adjusted by the pH of the solution in which the molecules aremaintained. For nucleic acids, the charge is generally negative anddetermined by the phosphate backbone, and is furthermore directlyrelated to the length of the nucleic acid. For the purposes of thepresent invention, this has certain disadvantages, since the size of thetarget 114 molecules can vary. Consider an application in which RNAmolecules associated with different genes will be measured. In suchcase, the length of RNA associated with each gene will vary according tothe length of the gene. Furthermore, RNA from higher organisms ispoly-adenylated, and the length of the “polyA” tail varies from RNA toRNA. This means that it is difficult to provide a relatively constantforce across all of the different RNAs, or even across RNAs associatedwith the same gene.

One method of overcoming this difficulty is to place an “electrophoretictag” on each molecule. The electrostatic charge of this tag will belarge compared with the charge of the polyA tail variation, andfurthermore can be substantial even with regards to the overall chargeof the RNA molecules. In this case, the variations of charge within RNAsassociated with a particular gene due to polyA tails will befractionally insignificant, and the charge differences between RNAsassociated with different genes will be fractionally small, even if theRNAs are of significantly different size, as long as the charge of theelectrophoretic tag is large enough.

FIG. 6 is a schematic diagram of an electrophoretic tag 270 in asandwich configuration. The electrophoretic tag 270 is generallycomprised of three functional components (or fewer components, of whichone or more components can comprise multiple functions). A tag bindingcomponent 272 binds the tag 270 to the target 114 through a means thatcan be either specific to the specific target 114 (e.g. a specificantibody or aptamer), or which can be common to a large number oftargets 114 (e.g. polyT, which will bind to polyA regions of mRNAs). Anindicator component 290 is detectable by a detector. An electrostaticcomponent 280 comprises a charged material, wherein the charge is largeand consistent from tag to tag. While the magnitude of the electrostaticcharge of the electrostatic component 280 can be broad within the spiritof the present invention, it is preferable for the charge to be at least1,000 net charges, and even more preferable for the charge to be atleast 5,000 net charges, and even more preferable for the charge to beat least 10,000 net charges. Furthermore, it is preferable for thecharge on the electrophoretic tag 270 to be of the same polarity as thecharge on the target 114. For example, for nucleic acid targets 114, itis preferable for the electrostatic component 280 to be negativelycharged.

It should be noted that at certain times, it can be convenient toindependently form an association between the electrophoretic tag 270and the target 114. That is, instead of associating the target 114 withthe probe 116, and then associating the tag 270 with the target 114, thetag 270 and the target 114 are first associated, where the associatedcomponent is called a tagged target 275.

The structure of the electrophoretic tags 270 can be quite varied withinthe spirit of the present invention. FIGS. 7A through F are schematicdiagrams of electrophoretic tags 270, showing differing arrangements ofcomponents to provide functionality within the scope of the presentinvention.

FIG. 7A is a schematic diagram of an electrophoretic tag comprised ofcross-linked DNA 281 as the electrostatic component 280 and fluorescentdyes 291 as the indicator component 290. The DNA is best largely doublestranded so that it interferes less with nucleic acid targets 114 andprobes 116, and is conveniently comprised of regions of double strandedDNA with single-stranded tails that interact with one another.Furthermore, it is preferable for the interacting regions to bechemically bonded to provide integrity to the tag 270 under a variety ofdifferent physical and chemical conditions. An example of this form ofelectrophoretic tag 270 is 3DNA (Genisphere, Hatfield, Pa.), which is adendromeric, cross-linked DNA structure which can be bound to bothfluorescent dyes as well as to a binding component 272. The bindingcomponent 272 is conveniently an antibody with specificity against thetarget 114, an avidin molecule with specificity to a biotin moietyattached to the target 114 (or conversely, a biotin moiety withspecificity against an avidin molecule attached to the target 114), anaptamer selected with specificity to a target, a nucleic acidcomplementary to a nucleic acid target 114, or other specific bindingcomponents. It should be noted that for use with messenger RNA targets,the binding component 272 is conveniently a polyT single-stranded DNAoligomer, which will bind to the polyA tails of the RNA, oralternatively a polyT Locked Nucleic Acid (Exiqon, of Vedbaek, Denmark)which has higher affinity for polyA than the unmodified polyT.

It should be noted that in many cases, the binding energy between thebinding component 272 and the target 114 will be chosen to be greaterthan that of the binding energy between the target 114 and the probe116. This can be arranged by either making the binding of the target 114to the probe 116 weaker, or more preferably, making the binding of thebinding component 272 to the target 114 stronger. One method to ensurethis is to create covalent links between the target 114 and the bindingcomponent 272. This can entail, for example, the incorporation of BrdUinto the polyT linker of the binding component 272, which can bephoto-activated to cause covalent links. In the case of proteins, if thebinding component 272 is comprised of a protein (e.g. an antibody), theprotein can be modified with photo-activatable cross-linking reagentssuch as aryl azides (e.g. phenylazide, hydroxyphenylazide, andnitrophenylazide) and after the target 114 is allowed to associate withthe binding component 272, light can be used to stimulate cross-linking.The unreacted cross-linking reagent can then be consumed using adeactivation reagent, which in the case of aryl azides can includereducing agents such as thiol-containing reagents.

While in most cases, the binding energy being discriminated is thatbetween the probe 116 and the target 114, it is also within the spiritof the present invention for the discrimination to take place regardingthe binding energy between the target 114 and the tag binding component272. Consider, for example, an antibody sandwich assay, in which boththe probe 116 and the tag binding component 272 comprise antibodies orparts of antibodies. In that case, it is equally useful for the weakerantibody-ligand binding energy—that is, the binding energy that is beingdiscriminated in the assay—to be with either antibody. This simplifiesthe design of such an assay, inasmuch as it is unnecessary to determinewhich of the antibody components to be used in the sandwich assay has astronger affinity for the target 114.

This ability to utilize both target 114-probe 116 binding energy as wellas target 114-tag binding component 272 is equally applicable to nucleicacids as well. Thus, the methods of the present invention will beeffective even if the target 114-tag binding component 272 associationis weaker than that of the target 114 to the probe 116.

Furthermore, this method still applies even if the association betweenthe target 114 and the tag binding component is not a specificone-to-one association. Consider, for example, the case where the tagbinding component 272 comprises a fixed length polyT oligonucleotide,which may be comprised of Locked Nucleic Acid nucleotides, whichassociates with the polyadenylated “tails” of messenger RNA. Thespecific association of probes 116 with their targets 114 can providethe spatial specificity of binding of the targets 114—that is, where inthe array 180 that the target 114 will bind—whereas the binding energybetween the target 114 and the tag binding component 272 can provide aconsistent binding energy that can be discriminated by the system.

It is also within the spirit of the present invention for covalentcross-linking to occur between both target 114 and probe 116, as well asbetween target 114 and tag binding component 272, so as to make acontinuously covalent linkage between the substrate and the indicatorcomponent 290. That is, given the incorporation of proper activatablecross-linking components into the probe 116 (see above for a discussionof activatable cross-linking reagents), after the reaction between thetarget 114 and the probe 116, activation of the cross-linking moietybound to the probe 116 can be performed, such that covalent cross-linksbetween the probe 116 and the target 114 are formed. Such reaction canoccur as well between the target 114 and the tag binding component 272,as described above. In such cases, the binding energy holding theindicator component 290 to the substrate is very large, so that specificbinding can be easily distinguished by its large binding force.

FIG. 7B is a schematic diagram of an electrophoretic tag 270 comprisedof an ionic polymer 282 as the electrostatic component 280 andupconverting phosphors 292 as the indicator component 290. The ionicpolymer can be conveniently linear or branched polyanion, with the ionicgroups comprising either carboxyl groups (if the pH of the buffer atwhich the tag 270 is to be used is near or above the pK of the carboxylgroup), or can also be a polyphosphate, polysulfate (e.g. polyvinylsulfonate, polystyrene sulfonate, sulfated starches, or dextransulfonate) or other polymer containing an inorganic acid moiety, whichcan comprise phosphates, quaternary amines, tertiary amines, secondaryamines, primary amines, sulfates, nitrates, and carboxylates. Theseionic polymers can be created via de novo synthesis from monomericreagents, or can alternatively be generated by modifications ofwell-characterized non-ionic or weakly-ionic polymers such as polyvinylalcohol or various starches. It should be noted that the highly ionicpolymers will be highly attracted to highly ionic species of theopposite polarity, and that therefore the electrostatic component 280needs to be tested to check for nonspecific binding to the substrate 120or other species in the analyte solution that can give rise to highbackgrounds in the detection assays.

Upconverting phosphors 292 are particles that convert lower frequencylight into higher frequency light (see Orasure Technologies, Inc. ofBethlehem, Pa.), and are convenient to use due to the few naturalcompounds having this property, leading to generally low background indetection assays.

FIG. 7C is a schematic diagram of an electrophoretic tag 270 comprisedof an ionic polymer 282 as the electrostatic component 280 and a directvisualization particle 293 as the indicator component 290. The particlecan be metallic (e.g. gold), ceramic, colored glass, or other opaque orlargely opaque material and is conveniently at least 250 nanometers, andmore preferably at least 500 nanometers, so that it is visible via lightmicroscopy. The ionic polymer 282 can be comprised of the same materialsas the ionic polymer 282 of FIG. 7B.

FIG. 7D is a schematic diagram of an electrophoretic tag 270 comprisedof an ionic polymer 282 in conjunction with a low nonspecific bindingpolymer 284 as the electrostatic component 280 and a light scatteringparticle 294 as the indicator component 290 of FIG. 6. The ionic polymer282 is similar to that shown in FIG. 7B. If this polymer 282 exhibitshigh nonspecific binding, it can be coated with a second polymer 284,such as a form of polyethylene glycol or polyacrylamide, which exhibitvery low nonspecific binding. This coating will in general involvecovalent bonding between the ionic polymer 282 and the low nonspecificbinding polymer 284.

The light scattering particle 294 can comprise a variety of materialsthat scatter light, including metals, ceramics and glass. The size ofthese particles is preferably smaller than 500 nm, and even morepreferably smaller than 200 nm and even more preferably smaller than 50nm. An example of such a light scattering particle 294 is resonancelight scattering particles by Genicon (San Diego, Calif.).

FIG. 7E is a schematic diagram of an electrophoretic tag 270 comprisedof a double stranded DNA molecule 285 as the electrostatic component 280and a quantum dot 295 as the indicator component 290. In this case, theelectrostatic component 280 is a linear, rather than a branched orcross-linked DNA molecule. The indicator component 290 and the bindingcomponent 272 are connected on either end of the DNA molecule 285. Thestructure can be assembled by attaching the binding component 272 to oneend of the single stranded DNA molecule, and then attaching theindicator component 290 to a complementary single stranded DNA molecule.As the two complementary single stranded DNA molecules hybridize withone another, the desired structure is generated. It should be noted thatthe double stranded DNA molecule 285 can be replaced with a singlestranded DNA molecule or with a linear polyionic polymer within thespirit of the present invention.

Quantum dots 295 function much in the same way as fluorescent dyes, butwith a considerably larger shift between the excitation and admissionfrequencies. This large shift allows the use of higher efficiencyoptical filters that reduce the amount of background noise in adetection assay. An example of quantum dots 295 is the nanocrystalsproduced by Quantum Dot Corp. (Hayward, Calif.).

FIG. 7F is a schematic diagram of an electrophoretic tag 270 comprisedof a linker component 274 linking double stranded DNA molecule 285 asthe electrostatic component 280 and a quantum dot 295 as the indicatorcomponent 290. The linker component 274 comprises attachment sites forthree components: the binding component 272, the indicator component290, and the electrostatic component 280. The linker component 274 willgenerally have three different binding groups which allow for selectivebinding of each group by the three components separately. An example ofsuch a linker 274 includes the amino acid cysteine, which has carboxyl,amino and thiol components of separable reactivities for synthesis, orserine, which has carboxyl, amino, and hydroxyl components. There are anumber of functional groups that can be used on the linker in order toallow it to interact with the components 272, 290 and 280. Thesefunctional groups can comprise, for example, thiols, aryl azides,alcohols, amines, epoxies, n-hydroxy-succinimide, biotin, avidin, orother chemically active groups or groups with high affinities (e.g.avidin and biotin).

It should be understood that in the preceding discussion of anelectrophoretic tag 270, the electrostatic component 280 and theindicator component 290 from the different examples can be combinedseparately to create tags of useful benefit. It is further understoodthat the electrostatic components 280 and indicator components 290discussed are not exhaustive, and any chemical or physical componentproviding similar function is within the present invention. Forinstance, the indicator component 290 can comprise many materials, suchas (and including modes of detection discussed above) enzyme indicators,chemiluminescent indicators, electrochemical (e.g. redox) indicators,radioactive indicators, and others types that are used in microarray,ELISA, and other biochemical and chemical assays, upconvertingphosphors, fluorophores, quantum dots, light scattering particles, lightabsorbing particles (e.g. colored particles), or phase contrastparticles (i.e. to confer index of refraction differences that can bevisualized in a phase contrast microscope or by surface plasmonresonance).

Many of these indicators can be used with optical detection means whichis matched to that of the indicator. Thus, for fluorophores, quantumdots, and upconverting phosphors, paired excitation illumination (e.g.laser excitation or broad-spectrum illuminators with bandpass filters)and emission-specific detectors (e.g. bandpass filtered) are utilizedalong with proper imagers (e.g. cameras with or without magnificationoptics). Light scattering particles will often use oblique incidentillumination (including standard darkfield condensers) or evanescentillumination, or may alternatively use phase contrast optics, sinceparticles with sufficient difference in refractive index to give rise tophase optical effects will also give rise to light scattering. Inaddition, the phase contrast particles will also generally be visible insurface plasmon resonance. Phase microscopy can be used for phasecontrast particles, and light absorbing particles and enzymaticreactions can be used in both phase contrast microscopy and brightfieldimaging (e.g. with microscopic imaging or other forms of magnification).Chemiluminescence can be detected with proper magnification anddetectors arranged to have the proper receptivity to thechemiluminescent signal. The descriptions above are not exhaustive, andother combinations of indicator and detector are within the spirit ofthe present invention.

It should also be noted that it is preferable that there be only asingle binding component 272 for each electrophoretic tag 270 so thateach target 114 is associated with only a single electrophoretic tag270. This can be handled by associating targets 114 with a largenumerical excess of electrophoretic tags 270 such that, on average, mostelectrophoretic tags 270 will be unassociated with target, and that mosttagged targets 275 will have only a single target 114.

The amount of charge on the electrophoretic tag 270 should generally becomparable to or greater than the charge on the targets 114. Forproteins, the charge may not be large, those nucleic acids in generalhave approximately one charge per nucleotide, and the size of thetargets can be hundreds to thousands of nucleotides (in a small numberof cases tens of thousands of nucleotides or more). While bacteria andother organism targets can have a large charge, there are also generallya number of places for the tag 270 to bind, and so the sum of many tags270 will often exceed the charge on the organism surface. In general, itis preferable for the electrophoretic tag 270 to have an averageabsolute net charge of greater than 1000, and even more preferablygreater than 5000, and most preferably greater than 20000.

It should further be understood that in most applications of the presentinvention the use of an electrophoretic tag 270 is not a requirement.That is, most targets 114 intrinsically comprise an electrostatic chargethat allows the target's 114 movement in electrostatic orelectrophoretic fields, and for which targets 114 the tags do notrequire an electrostatic component. It is within the spirit of thepresent invention, where the term electrophoretic tag 270 is used inthis description, that a non-electrophoretic tag can be used inconjunction with the naturally occurring electrostatic charge on thetarget 114. Furthermore, the charge of these molecules can often beadjusted by pH, and it can be convenient to adjust the pH at whichelectrophoresis occurs to alter the electrostatic charge on the target114.

Competitive Assay Formats

The assay formats described above related primarily to sandwich assayformats. However, in the case of very small targets 114, such ashormones or drugs of substance abuse, it is difficult to find reagentsthat allow simultaneous, high-affinity binding of both a probe 116 and atag binding component 272. Without two such binding reagents, thesandwich assay is performed with difficulty.

An alternative is a competitive assay, in which a specific binding probe116 to the target 114 is immobilized, as before, on the substrate. Addedto the analyte containing the target 114 is a competitor, which binds tothe probe 116 with similar affinity to that of the target 114, and towhich is covalently bound an indicator 290. In the absence of target 114in the analyte, a given amount of the competitor will bind to the probe116. However, if the analyte contains the target 114, the binding of thecompetitor will be reduced. Thus, in the competitor assay, the target114 is not directly detected, but rather its abundance is evidenced bythe reduced binding of the competitor.

The competitive assay format is used advantageously in the presentinvention, given the requirements for consistent and reproduciblebinding, which is improved by the reaction acceleration of the presentinvention. Furthermore, because the present invention uses relativelyshort reactions, as well as rapid washing, relatively low affinityprobes 116 can be used that would otherwise lead to loss of signal withconventional washing and detection methods. Note that this latteradvantage accrues not only to competitive assay formats, but sandwichassay formats, as well.

Attachment of Probes

As mentioned above, the probe 116 is attached to the substrate 120through a linker 118. This linker 118 conveniently comprises a coatingwith functional groups, wherein the functional groups permit the bindingof the probes 116. Also, the coating preferably has low nonspecificbinding, so that target 114 or indicator 290 in solution that is notspecific for the probe 116 does not bind to the surface. Examples ofsuch coating materials include Codelink by Amersham and OptiChem byAccelr8, which comprise hydrogel-like coatings with both very lownonspecific background, as well as electrical properties. Alternatively,the coating can comprise a derivatized silane.

Other Components

There are a number of other components comprising compete systemsaccording to the present invention, including power controllers forestablishing the potential differences between electrodes that will because and control the electrophoretic force on the targets 114,illuminators to illuminate the indicators 290, detectors to detect thesignals generated by illumination of the indicators 290, and storagecontrollers (e.g. controllers and hard disk drives) that store theinformation from the detectors and then present it to the user orcompare information from multiple sources or times. Some of thesecomponents are well-known in the art, such as electrophoresis powersupplies (which can be computer controlled and which can be set toprovide either constant voltage or constant current, and which can besupplemented with digital or analog electronic circuitry to provide lowto high frequency waveforms as described elsewhere in this specificationand which can also be used for dielectrophoresis), illuminators (e.g.lasers, arc lamps, incandescent lamps, microscope light condensers, andwhich can involve methods of coupling the light into light waveguides),indicators (as described above and below), detectors (cameras, lenses,filter sets, image analysis software), and the like, even as theirarrangement and use is novel and to novel effect in the presentinvention. Where the components differ from prior art, they will bediscussed both above and below.

Functional Description of the Present Invention

The present invention can be considered to comprise three steps as shownin FIG. 8, a schematic block flow diagram of the steps of the presentinvention.

In a first step 300, the sample comprising the target 114 is preparedfor use in the assay. The method of preparation depends upon the type ofmaterial being assayed, and can include the maceration of solid tissue,or alternatively the lysis of cells if the material to be assayed is ofintracellular origin. Solid material can be removed from the preparationby centrifugation, filtration or other means, and if nucleic acid is thetarget 114, the nucleic acid can be purified away from the rest of thestarches, lipids, and proteins of the preparation (indeed, whatever thenature of the target 114, it can be convenient to remove components thatmay interfere with later stages of the analysis). If the material isnucleic acid, it can be amplified by means such as polymerase chainreaction (PCR) or rolling circle amplification or other amplificationmethods. Generally, the material should be maintained in a conditionthat preserves target 114 reactivity with the probe 116, as well as thereactivity of the electrophoretic tag 270 with the probe 116. Ingeneral, the least amount of preparation will be used that allows forboth high signal and low background, due to the cost, time, andartifacts that are generally introduced via preparation.

In this preparation step, the electrophoretic tag 270 can be reactedwith the target 114 in order to generate a tagged target 275 asdescribed in FIG. 6. Alternatively, this reaction can occur later in theprocess as described below.

In a second step 310, the tagged target 275 and the probe 116 arereacted. In the case of nucleic acids, this can comprise a step ofhybridization. In the case of protein targets 114, this can comprise anantibody-hapten reaction, a protein-aptamer reaction, or aprotein-protein reaction.

It is a teaching of the present invention to accelerate the reactionbetween the tagged target 275 and the probe 116. If the reaction isincomplete, the amount of target 114 bound to the probe 116 will be lessthan optimal. Additionally, because the rates of reaction for differenttargets to different probes 116 are generally different, and because theamounts of target bound to the probe will not be linear with time, it ishard to quantitate the amount of target 114 bound to the probe 116without the reaction having gone to completion.

The means of accelerating the reaction involves the movement of thetagged target 275 under the influence of an externally applied force,which can be conveniently an electrophoretic, dielectrophoretic ormagnetostatic force. For this description, electrophoretic forces willbe used as an example. This force can be applied either by the placementof an electrode 200 under the positions of the probe 116, or through theinfluence of electrodes 210 and 220 that are placed to the sides of theprobe locations 170, in a manner to be described below.

In the third step 320, unreacted tagged target 275 is separated from theprobe 116 and the tagged target 275 that remains attached to the probe116 is detected. Importantly, conditions are set such that tagged target275 that is properly reacted with the probe 116 is not removed, and thatother tagged target 275 that is nonspecifically bound to the probe 116or to the substrate 120 is removed. It should be appreciated that withmultiple tagged target 275 and probe 116 pairs, the binding force willbe different in the case of each pair. In order to discriminate specificfrom nonspecifically bound material, a different discriminating forcewill optimally be used for each probe 116. This methodology is outlinedin FIG. 9, a graph of the amounts of material bound versus the bindingenergy. Line 340 represents the amount of nonspecifically boundmaterial, and is characterized by a very large amount of material thatis loosely bound, a variable amount of material bound with theintermediate energy, and some amount of material which is boundstrongly. It should be noted that the shape of this curve will bedifferent depending upon the materials being assayed, and that thearguments made below are not dependent upon the particular shape of thecurve.

Two targets are shown in the figure: target X is represented by line 344and target Y is represented by line 346. Target X has a lower bindingenergy with its corresponding probe 116 than the target Y. In aconventional assay in which a single discriminating wash is used for alltarget-probe pairs, the discriminating energy must be chosen such thatit is less than the binding energy of the least tightly bound target.This binding energy is represented by dashed line 342. It can be seen,however, that using a single discriminating energy results in abackground represented by the total of all nonspecific binding 340 tothe right of the line 342. In the location of target Y, for instance,significant nonspecifically bound material with binding energy both lessthan and greater than that of the specifically bound target Y will bepresent.

In the present invention, washings corresponding to a number of bindingenergies will be used. These binding energies are represented by dashedlines 350 at forces represented by lines A, B, C, D, and E, which aresuccessively applied. For instance, a first “wash” at discriminatingenergy A is applied, and virtually all of the tagged material bound atthe location of probe X is detected. Then, washing at discriminatingenergy B is applied, and the material bound at the location of probe Xis once again determined. The difference between the material at wash Aand wash B is considered to be specifically bound material correspondingto target X. After subsequent washings at discriminating forces C, D,and E, the amount of target Y is considered to be that material presentin wash D and not present in wash E at the location of probe Y. Thus,the proper discriminating energy for each target-probe pair is utilizedusing a bracketing pair of discriminating washes. In each case, thenonspecific background is only that part of line 340 that falls betweenthe pair of discriminating washes specific for that probe location.

It should be noted that the rupture force is dependent on applied forceand the rate of force applied. For example, under non-equilibriumconditions, the rate of force applied per unit of time actually changesthe width of the potential energy landscape effectively increasing theintegrated energy (force through applied through a distance) required torupture the interaction. Another way of stating this is that rapidpulling apart does not allow time for the relatively slower unbindingprocess, so a large force is required to rip apart the molecules andthat the energy landscape or barrier is significantly higher at rapidloading over lower loading rates of force. So depending on force loadingrate, there will be multiple critical rupture forces. Furthermore, theshape of the loading rate versus critical rupture force is different foreach receptor ligand interaction, since the intermolecular interactionsare different. Thus, multiple antibody-antigen interactions and nonspecific binding can be resolved with dynamic force analysis—that is, byobserving the rupture force plotted against loading rate, overlappingbinding energy curves can be separated depending on loading rate.Therefore, the shape of the applied voltage curve is very important tocontrol.

It should be noted that the reaction step 310 and the washing anddetection step 320 can be performed cyclically multiple times. That is,after the washing and detection step 320 has removed all of the target114 from the probe 116 (or the tag 270 from the target 114), anothercycle of reaction and washing/detection can take place. This has twoprimary advantages. Firstly, if there are a small number of targets 114in the analyte, the number of binding events detected will be small. Byrepeating the reaction and washing/detection steps, a larger number ofbinding events can be counted, improving the statistics of the results.

Furthermore, differing voltage dynamics (for example, voltage rampprofiles) can be utilized in each cycle of the two steps 310 and 320, inorder to distinguish specific from nonspecific binding events that mightbe distinguished in only by differing responses to voltage dynamics. Forexample, in a first cycle, the voltage dynamics can involve a stepfunction in which voltages are changed rapidly, whereas in a secondcycle, the voltage dynamics can involve a slow, ramped increase involtage.

It should be noted that in order for the foregoing methods to be used, ameans of real-time detection of the tagged target 275 to the probe mustbe available. That is, if each wash were to take a considerable amountof time and require many manipulations, only a small number of differentdiscriminating washes could be used. With a real-time detection method,however, a large number of discriminating washes can be implemented,getting better definition of specific versus nonspecific bound material.

It should be further noted that in the following discussion, the use ofelectrophoretic forces can be used in both accelerating the reaction aswell as in providing discrimination between specific and nonspecificallybound material. It should be understood, however, that it is within thespirit of the present invention that in a given application, both usesof the electrophoretic forces acting on target-probe complexes, oralternatively, only one or the other of these uses of electrophoreticforces can be used to beneficial effect.

The number of discriminating washes used for a given assay can depend onthe specific target-probe pairs used, but in most cases, the number ofwashes is preferably less than two times the total number of targets 114being detected (with two steps each to “bracket” a particular target114). It is also convenient for the spacing of the discriminatingenergies not to be evenly spaced, but to be tuned to bracket individualor groups of target-probe binding energies. It is also within the spiritof the present invention for the wash to be performed as a continuousgradient of stringency, which can be linear in stringency versus time,or non-linear, with detection of the target 114 being performed atintervals, wherein the number of detections is preferably less than twotimes the total number of targets 114 being detected.

Because of the natural dissociation constant for each of thetarget-probe pairs, which relates to a stochastic dissociation that isoften thermally driven, it is convenient to choose conditions fordiscriminating washes in which this statistical component ofdissociation is most attenuated. These conditions will in the case ofnucleic acids, for example, involve moderate pHs, low temperatures, andhigher salt concentrations. These conditions for proteins might includeionic strength, pH gradients, hydrophobicity, and solvent polarity aswell.

It should be noted that while the present invention teaches the use ofelectrophoretic potential for discriminating washes, one aspect of thepresent invention relates more generally to the realtime detection ofvarying washing regimes, wherein the washing regimes can comprise avariety of different physical and chemical conditions beyondelectrophoretic force. These forces can comprise increasing temperature,magnetic field strength (should the tagged target comprise aparamagnetic particle), dielectrophoresis (for particles, bacteria, andother targets and tagged targets), shear fluid flow, ionic strength(either increasing or decreasing), pH (either increasing or decreasing),surfactant concentration (either ionic or non-ionic), or competitorconcentration (e.g. if the target 114 is a protein, to add increasingamounts of that protein so that when the bound target 114 is released,the competitor preferably binds to the probe due to its highconcentration). In addition, more than one of these conditions can beapplied either simultaneously or in sequence. While it is generallypreferable for these conditions to be applied with gradually increasingstringency, it is also within the spirit of the present invention forthe stringency to be increased in a step function, with rapid discreetincreases in stringency. By monitoring the binding of the target 114 tothe probe 116 at various increased stringencies of any of theseconditions, discrimination of specific from nonspecific binding can beimproved.

Function Involving Electrodes not Underlying Probe Locations

FIG. 10 is a schematic block flow diagram of a system involvingelectrodes not underlying probe locations. In a step 360, a negativelycharged target 114 is added to a reaction cell similar to that shown inFIG. 4B. For purposes of this discussion, the target 114 will beconsidered to be a nucleic acid, and the probe 116 will be considered tobe a complementary nucleic acid sequence, although in practice, theprobe 116 could also comprise proteins, glycoproteins, starches, orother molecules of interest. In a step 362, the electrode A will bepositively biased relative to electrodes B, P, Q, and R, attracting thenegatively charged target 114 to that electrode, on which it willcollect. In a step 364, electrode B is positively biased relative toelectrodes A, P, Q and R, so that the target 114 is drawn to theelectrode B, during which transit it is brought into close proximitywith the probes 116 at locations 170 in array 180, so as to facilitatereaction between the target 114 and the probe 116 can take place. Thenegative bias on the electrodes P, Q, and R maintains electric fieldvectors with downward pointing components during movement of the target114 so that the target maintains close proximity with the probes 116.Once again, it should be noted that the location and the relativevoltages on the upper electrodes P, Q, and R can be adjusted to shapethe electric field vectors in the cell. The magnitude of the electricfield vectors upward and lateral from the positions 170 must be lowerthan the binding force that binds the specifically bound targets 114 totheir corresponding probes 116.

In an optional step 366, weakly-adhered nonspecifically-bound materialcan be removed from the array 180 by placing a small net positive biasto electrodes P, Q, and R, drawing the material away from the array 180.In a step 370, an event indicator, for example an electrophoretic tag270, is added to the cell. Because of the high concentration of theelectrophoretic tag 270, reaction with the target 114 occurs rapidly. Inaddition, reaction of the electrophoretic tag 270 with the target 114can be accelerated by electrophoretic means. In a step 372, theelectrode A is positively biased relative to electrodes B, P, Q, and R,transporting the electrophoretic tag 270 to the electrode A. In asubsequent step 374, the electrode B is placed positively biasedrelative to electrodes A, P, Q, and R, moving the electrophoretic tag270 from electrode A to electrode B, with generally downward pointingelectric field vectors, so that the electrophoretic tags 270 are inclose proximity to the targets 114, with which they react. In a step376, the electrode B positive bias is increased in a generally stepwisefashion as the amount of material bound at the probe locations 170 ismonitored in order to determine the material that is specifically boundand to discriminate it from material that is nonspecifically bound. Itshould be appreciated that the event indicator can be a tag withoutparticular electrophoretic properties, should the target 114 be itselfcharged. Furthermore, there may be no event indicator should the target114 itself have properties of fluorescence, light absorption, indexdifference with the medium, or other properties such that it isdetectable, rendered the step 370 optional.

It should also be noted that the targets 114 can be made to move backand forth multiple times between the electrodes 210 and 220, in eachcase increasing the amount of target 114 that binds to the probes.

Function Involving Electrodes Underlying Probe Locations

FIG. 11A is a schematic block flow diagram of the operation of a cellinvolving electrodes underlying probe locations, and can be bestunderstood in relation to FIG. 4A. In the step 360, a negatively chargedtarget is added to the cell. In a step 382, the electrode D ispositively biased relative to electrode C, causing the target to migrateonto the electrode where it is in close proximity to the probe 116placed on array locations 170 on the array 180. Because of the closeproximity, the reaction between the target 114 and the probe 116 occursvery rapidly. In a step 370, an event indicator, for example anelectrophoretic tag 270, is added to the cell. In a step 386, electrodeD is once again positively biased relative to electrode C. Under theinfluence of the electric field, the electrophoretic tag 270 migrates toelectrode C wherein it reacts with the target 114. In a step 388,electrode C is set at a positive biased relative to electrode D.Electrode C's positive bias is increased in a generally stepwise fashionas the amount of material bound at the probe locations 170 is monitoredin order to determine the material that is specifically bound and todiscriminate it from material that is nonspecifically bound.

It should be appreciated that the step 370 and the step 386 can beeliminated by adding the electrophoretic tag 270 to the target 114 priorto adding the target 114 to the cell in the step 360. In this case, thetarget 114 is converted to a tagged target 275 prior to the applicationof a positive bias on electrode C in the step 382. The creation of thetagged target 275 can occur within the cell as shown in FIG. 11B, aschematic flow diagram of the operation of a cell involving electrodesunder the probe locations using a tagged target 275. In the step 360,the negatively charged target is added to the cell, and in the step 370,electrophoretic tag 270 is additionally added to the cell. At thispoint, a tagged target 275 is generated. In the step 382, the electrodeD is positively biased relative to electrode C and the tagged target 275moves into close proximity with the probes 116 at the target locations170, where reaction with the probes 116 occurs. In the step 388,discrimination of specifically bound versus nonspecifically boundmaterial is performed as before.

The monitoring of the binding of the target 114 that occurs in the step388 can be performed for an average of all of the material that isbound—for example, measuring the total output of light that is scatteredfrom a tag that has a light scattering indicator. However, if thedetector is an optical detector, and the detector is an imaging detectorsuch as a camera or a laser scanner coupled with a photo multipliertube, it is also within the spirit of the present invention for thebinding to be determined for individual targets 114. In this case, thedetector will need to store the locations of each target 114 betweensequential detections, and the strength of binding of each target 114 toeach probe 116 can then be determined.

Use of Magnetostatic Forces

It should be noted in the discussions above that magnetostatic forcescan be substituted in certain cases for electrostatic forces. For theuse of magnetostatic forces, however, the targets 114 must be taggedwith paramagnetic particles, given that for the most part, thebiological molecules or organisms to be detected are not in themselvesmagnetic. Examples of such particles include Estapor particles fromBangs Laboratories (Fishers, Ind.), and Dynabeads from Dynal, Inc.(Norway). Thus, the particles 293 and 295 of FIGS. 7C, E and F would besubstituted with paramagnetic particles, which are preferably less than1 micron in diameter, and more preferably less than 250 nm in diameter,and most preferably less than 100 nm in diameter; in general, thesmaller the particle, the less it interferes with the diffusion of thetarget 114 towards the probe 116, and the faster the reaction kinetics.Instead of electrodes, the placement of permanent or electromagnetseither above or below the probe 116 (in relation to the substrate 120)provides the force that moves the magnetically tagged targets 114towards or away from the probe 116. The magnitude of this force can beadjusted either by changing the distance of the magnetic field sourcefrom the probe 116 or the placement of shims of differing magneticpermeability, or in the case of an electromagnet, adjusting the currentthrough the coils, the physical distribution of the coils, the presenceof magnetically permeable material in or around the coils, and othersuch means as known in the art.

Real-Time Detection

As described above, the use of multiple washes of differingdiscrimination, as well as the monitoring of the binding of the targetto the probe require the use of real-time monitoring. This is to bedistinguished from the common conventional situation wherein after thereaction has proceeded for a predetermined period of time, the reactionis completed, the washes are performed, and then the substrates on whichthe reaction was performed are then prepared for detection. In many ofthe preferred embodiments of the present invention, an optical means ofdetection is employed. In those instances where the electrodes areopposed to each other (e.g. parallel and opposite), in order for opticaldetection to take place, one or both of the electrodes is preferablyoptically transparent, in order for an external optical device toreceive the optical signal that is generated between the two electrodes.

With reference to FIG. 4B, this is easily accommodated, wherein thesubstrate on which the array 180 is placed can be transparent. However,it may be preferable for the detector in that case to be placed abovethe electrodes 195P, Q and R, or alternatively in the case of anarrangement such as FIG. 4A, the detector will generally be external tothe electrodes 190 and 200. In such cases, the use of opticallytransparent electrodes is preferred, for which the preferred materialfor these electrodes is indium tin oxide (ITO). Because ITO is notstable generally to voltages above 2 V, this means that the potentialbetween the electrodes in the case of ITO should be preferablymaintained below this potential, as will be described in more detailbelow.

A more general discussion of detection will be provided below.

Control of Reaction Acceleration

The acceleration of reaction according to the methods above can beimproved by varying the electric fields both spatially and temporally soas to improve the reaction of the probe 116 with the target 114. FIG.12A is a schematic diagram of three electrodes arranged on twoperpendicular axes within a reaction cell. Electrode E1 and electrode E2are representative of electrodes 195, while electrode E4 isrepresentative of an electrode 200. That is, the array 180 is placed ontop of the electrode E4. The voltage potential between the differentelectrodes will be varied in such a way so as to improve the reactionrates as described below. In the discussion below, the use of the termstarget 114 and tagged target 275 are used roughly interchangeably. FIG.12B is a graph of the potential difference between the electrodes E2 andE4 as they vary with time, with electrode E4 biased positively to E2.There are three time periods represented on the graph, denoted as timesT1, T2 and T3. In the period T1, the voltage potential is maintained fora considerable period, such that the majority of the target 114 isbrought into juxtaposition to the probe 116. Because of the potentialdifference, both the probe 116 and the target 114 can be forced downwardonto the substrate 120, wherein the maintained voltage restricts theirability to react with one another. This will occur when the probe 116and the target 114 are of the same polarity of electric charge, such asin the case of nucleic acid hybridization, although in othertarget/probe pairs (e.g. protein-protein interactions), the chargepolarity can be different in the target 114 and probe 116. Even in suchcases, the target 114 can then be electrophoresed beyond the probe 116,impeding the reaction between the probe 116 and target 114. In bothcases, it is convenient to have a period, described below, that reversesor relaxes the effects of the electrophoresis.

In the period T2, the voltage potential can be removed allowing freemovement of the target 114 and the probe 116, accelerating the rates ofreaction. However, during this period T2, the target 114 is allowed todiffuse away from the probe 116. Thus, during the period T3, the voltagepotential is once more applied to maintain the close proximity of thetarget 114 in the probe 116. The periods T2 and T3 can be cyclicallyrepeated, until such time that the majority of the complementary target114 and probe 116 are reacted. The durations of the various periods canbe varied depending upon the topology of the reaction cell, thecharacteristics of probes 116 and targets 114, as well as the variouselectrostatic charges on the different components, and the manner inwhich probe 116 is affixed to the surface 120. In general, for largervertical and lateral dimensions of the cell, period T1 will be larger toallow for the larger distances over which the target 114 must be moved.

FIG. 12C is a graph of the potential difference between the electrodesE2 and E4 as they vary with time, arranged alternatively to that in FIG.12B. Again, as in FIG. 12B, during initial period T1, the target 114 isallowed to migrate under the influence of the electrophoretic force tothe probe 116. In this case, during a period T4, the electric field ismaintained at a very low level so as to maintain the juxtaposition oftarget 114 to probe 116, but with a lower force than that used in FIG.12B. This lower force is used in order to allow more movement of boththe target 114 and the probe 116 so that they are not topologicallyconstrained during the reaction. During an optional period T5, theelectric field can be reversed very mildly, so as to release any target114 that may have become enmeshed on the surface 120. The relativeduration of the periods T4 and T5 will depend upon the number offactors, including the type of surface to which the probe 116 isattached, the charge of the electrophoretic tag 270, the binding forcebetween the target 114 in the probe 116, the physical size of theelectrophoretic tag 270, and other factors. It should also be noted thatthe duration of the successive periods T4 or the successive periods T5need not be equal and may change over time.

FIG. 12D is a graph of potential differences between spatially displacedelectrodes, such that the electric field changes not only magnitude butalso in direction. With reference to FIG. 12A, electrode E2 is nearlyvertically displaced (i.e. directly opposed) from the electrode E4, andthe electrode E1 is both vertically and laterally displaced from theelectrode E4. As can be seen from the graph, electrode E4 is alternatelybiased positively and negatively relative to the vertically-displacedelectrodes E1 and E2. In addition, in certain cases, the bias isrelative to E1 and in other cases the bias is relative to E2. Thiscauses the electric field to vary in polarity, in magnitude, and indirection. This variation in direction means that tagged targets 275that become sterically trapped on the surface 120 will feel force invaried directions that can facilitate in releasing them from theirentrapment.

This arrangement can be carried out with various topologicalarrangements. FIG. 13A is a schematic diagram with three electrodes 195displaced in two dimensions over a single electrode 200. Electrodes E1and E3 are displaced in perpendicular directions from the electrode E2which is vertically displaced from the electrode E4. FIG. 13B is a graphof potential differences between the electrodes of FIG. 13A. ElectrodeE4 is maintained at a roughly constant positive potential. The otherelectrodes, however, cycle between a nearly neutral potential and anegative potential, causing the electric field to cycle in directionwith relatively constant magnitude.

It should be noted that the topological arrangements of the electrodesE1, E2, and E3 will vary with the shape of the cell. For instance, ifthe cell is between a microscope slide and a cover slip, the thicknessof the cell can be measured in hundreds of microns, which would cause anelectric field between the electrodes E1 and E4 to be nearly horizontal.If the cell is within a microtiter well, the depth of the cell will becomparable to that of its width, such that the electric field betweenelectrodes E1 and E4 will be more nearly vertical.

Electrochemistry to Improve Electrophoretic Acceleration

The electrophoretic reaction acceleration can be performed in a normalbuffer, using the electrolysis of the water or the constituent salt ions(e.g. sodium and chloride) to engage in redox reactions at theelectrodes as required to provide the current for the electrophoresis.There are a number of difficulties associated with the use of thesebuffers, however, for which we will use sodium chloride as an example.Firstly, if indium tin oxide (ITO) or other redox active materials isused at one or both electrodes (e.g. to provide an optically transparentor translucent, conductive electrode), the redox potentials powering theelectrophoresis need to be less than that at which the electrode willparticipate in redox reactions. In the case of buffers with sodiumchloride, for instance, the potential at which redox reactions occur athigh rates is greater than 2 Volts, at which potential the ITO isunstable.

Furthermore, the redox products of sodium chloride electrochemistryinclude Na metal which reacts in water to form the strong base NaOH, andCl₂, which reacts with water to form strong oxidizing reagents. Thesereagents, being very active, may be deleterious to the targets 114 andtags 270 being electrophoresed towards the electrodes.

Also, while salt provides conductivity to the electrophoresis, it alsocompetes with the charged material being moved—the larger theconductance of the buffer, the lower electrophoretic force that isencountered by the material. Thus, it is beneficial to limit theconductance of the buffer. In general, it is preferable, therefore, forthe conductivity of the buffer to be less than 1 mS/cm, and even morepreferable for the buffer to be less than 100 μS/cm, and even morepreferable for the conductivity of the buffer to be less than 100 μS/cm.In many instances, it is important for the ionic strength of the buffer,however, to be maintained at some reasonable level (e.g. >10 mM), forexample, for the viability of cells or to preserve the reaction ofproteins or nucleic acids (e.g. hybridization), or alternatively to havea buffer to maintain a pH range. In these cases, it is convenient to usezwitterionic molecules to maintain ionic strength or pH. Specifically,in the case where nucleic acid hybridization is desired, it isconvenient to use histidine buffer (e.g. see U.S. Pat. No. 6,051,380).

Choosing Appropriate Redox Accelerants

In order to reduce these effects, it is preferable to provide redoxagents that do not suffer from the problems listed above. An example ofsuch reagents is the benzoquinone/hydroquinone system. In this case,hydroquinone is oxidized at the anode to benzoquinone, and benzoquinoneis reduced at the cathode to hydroquinone. Because the reactions arecomplementary at the electrodes (i.e. have reversed potentials), theonly cell potential is due to differences in concentration rather thandifferences in standard potential at the electrodes, and thus theelectrophoresis redox reaction occurs at relatively low potentialsbetween the two electrodes. Furthermore, because the two species are notcharged, the redox agents do not significantly increase the conductivityof the solution and thus do not compete with the charged molecules (e.g.DNA) or material (e.g. bacteria) for transport via electrophoresis.

The redox scheme as described above can operate either with respect to aclosed or open system. FIG. 14A is a schematic diagram of a closedsystem for electrophoresis. On an upper substrate 870 is a cathode 850,and on a lower substrate 870 is an anode 860. In the region between theelectrodes are two compounds: an oxidized molecule (OX1) which accordingto the discussion above could be benzoquinone, and a reduced molecule(RED1) which according to the discussion above could be hydroquinone. Atthe cathode 850, OX1 is reduced to RED1, which then moves either byelectrophoresis or by diffusion to the vicinity of the anode 860. At theanode 860, RED1 is then oxidized to OX1, which then moves either byelectrophoresis or by diffusion to the vicinity of the cathode 850,where the cycle can repeat itself.

Depending on the amount of availability of charge carriers (which can beunrelated electrolyte, RED1 and/or OX1, or charged molecules ormaterials to be transported), the electrophoretic force, and thereforethe rate at which molecules or materials can be transported, can belimited to the rate of diffusion of OX1 to the cathode and RED1 to theanode. This rate of diffusion can be improved significantly be makingthe distance between the cathode 850 and the anode 860 small—it ispreferable for this distance to be less than 2000 microns, even morepreferable for this distance to be less than 1000 microns, and even morepreferable for this distance to be less than 500 microns.

The system of FIG. 14A is closed, in that the system can be closed offfrom the environment, and electrophoresis can be continued indefinitelywithout replenishing the redox reagents. FIG. 14B is a schematic diagramof an open system for electrophoresis. The arrangement of substrates870, cathode 850 and anode 860 is the same as that of FIG. 14A. However,in this case, there are two pairs of reagents which do not regenerateeach other (either directly as in benzoquinone and hydroquinone, or bymutual quenching of redox products, as described below). At the cathode850, an oxidized molecule OX3 is reduced to the molecule RED3, while atthe anode 860, a reduced molecule RED2 is oxidized to OX2. The productsRED3 and OX2 do not react with one another to regenerate the reactants,and so as soon as OX3 and RED2 are exhausted, electrophoresis willterminate. Thus, in this open system, in order to maintainelectrophoresis, the reactants OX3 and RED2 must be continuouslyreplenished, which is accomplished generally by maintaining a flow ofnew reactants in the electrophoresis buffer into the space between theelectrodes. It should be noted that this flow will also remove anytargets 114 to be transported, unless such targets 114 are somehowimmobilized to the cathode 850 or anode 860 by the time that the bufferexits the region between the electrodes 850 and 860.

There are numerous redox pairs that can operate within the presentinvention. As described above, benzoquinone and hydroquinone are wellsuited to this, and are preferably used in concentrations above 1 mM,more preferably used in concentrations above 10 mM, and mostconveniently used in concentrations above 30 mM. It should be noted thatthe use of benzoquinone and hydroquinone are limited to an extent bytheir limited solubility, and so more polar or charged derivatives canbe conveniently used to increase their solubility, such derivativesincluding the substitution of the ring carbons not bonded to carbon withhalogens, nitrates, hydroxyls, thiols, carboxylates, amines, and othersuch moieties. It should be noted that it is optimal for the system forthe resulting redox agents to be uncharged (except as will be shownbelow), so that their distribution is not affected by the systemelectrophoresis, and so the substitution with a positively charged group(e.g. an amine) is balanced by a second substitution with a negativelycharged group (e.g. a carboxylate), such as in 2-amino, 5-carboxypara-benzoquinone. In such cases of derivatized benzoquinones andhydroquinones, the concentrations of the redox reagents can beconveniently increased.

Other similar redox pairs include ketone/alcohol and aldehyde/alcoholpairs, whose ketone carbonyl group can be flanked by alkyl or arylgroups, which groups can also be derivatized with halogen, nitrate,hydroxyl, thiol, carboxylate, amino and other groups so as to modify thecharge on the molecule or to increase its solubility. Another convenientsystem is that of dithiothreitol/dithioerythritol and their oxidizedforms (which can be formed by the partial oxidation of solutions of thereduced forms, for example, by hydrogen peroxide), or alternatively byalkanes with terminal thiol groups (e.g. 1,5 dithiobutane). In general,it is preferable for the two thiol groups to be on the same molecule (asin dithiothreitol) as opposed to on separate molecules (e.g. as inbeta-mercaptoethanol), so that the oxidation reaction is a unimolecularreaction that is relatively less sensitive to concentration (althoughthe single thiols, such as beta-mercaptoethanol, are acceptable reducingagents for many applications).

It should be noted that the redox pairs above are oxidized and reducedin pairs of electrons in such a manner that the charge on both redoxpairs is the same, and is preferably neutral. The requirement that pairsof electrons be transferred can, however, reduce the rate of thereaction, and so it can also be convenient to use pairs in which oneelectron is transferred in the redox reaction. Examples of such pairsinclude ferrocene/ferrocinium and their derivatives, andferrocyanide/ferricyanide. In such cases, it is preferable to use pairsin which the reduced product is neutrally charged, and the oxidizedproduct is positively charged in those cases where negatively chargedmolecules or materials will be transported. The reason for this is thatthe oxidized product supplies countercharge to the transport of thenegatively charged transported molecules, and the reduced product isuncharged, and so does not compete for transport with the negativelycharged transported molecules.

Another configuration of the system is that where the products of theredox reactions quench one another, such as in the following:Anode: 2I⁻2e ⁻→I₂Cathode: S₄O₆ ⁻²+2e−→2S₂O₃ ⁻²

The products of this reaction spontaneously react with one anotheraccording to 2S₂O₃ ⁻²+I₂→S₄O₆ ⁻²+2I⁻, regenerating the starting state.The use of iodide or another halide is convenient, since the iodide ismoved through electrophoresis towards the anode, and the resultingiodine is neutrally charged and can move through osmosis towards theother electrode where it will meet with the thiosulfate for theregeneration of the initial system.

In open loop systems without recycling, where the redox pairs do notregenerate one another during their respective reactions, the range ofredox agents is broader, and conveniently includes compounds includingglutathione, ascorbate, methyl viologen, phenazine methosulfate, trolox,and others, including their redox pairs (such as GSSG for glutathioneand dehydroascorbate for ascorbate, oxidized methyl viologen for methylviologen). In this case, it is sometimes convenient that the charge ofthe molecule be such that the reactant be attracted towards theelectrode at which it will participate in redox reactions (i.e.reactants to be oxidized at the anode should be negatively charged andreactants to be reduced at the cathode should be positively charged).This can generally be accomplished by derivatizing the molecule with oneor more appropriately charged moieties. The main disadvantage of this isthat a negatively charged redox agent, while increasing the rate ofreaction, can also compete with the negatively charged transportmolecules, such that increasing the amount of redox reactant can evenreduce the overall transport of the transport molecules. Thus, careneeds to be taken through experimentation to ensure that negativelycharged redox reagents do not have an overall deleterious effect.

It should be noted, however, that small molecules of a redox pair,because of their high diffusion rates, are only moderately affected bythe electrophoresis, and over the short distances that generally existbetween the cathode and anode, show a modest gradient over theelectrodes (often only 2-3 fold, and generally less than 10-fold). Inthis case, it may be useful to have one or both redox reagents beneutral or positively charged. In the case where both agents arepositively charged, it is preferable that the agent that reacts at thepositively charged anode be in larger overall molar concentrations tocompensate for the lower local concentrations at the anode.

In those cases where microorganisms are being transported in thepresence of redox agents, it is important to note that some of the redoxagents mentioned above can have toxicity for microorganisms. In caseswhere the subsequent growth or monitoring of live organisms is desired,this can be a significant problem. For that reason, it is useful eitherto use low concentrations of the toxic redox reagent (generally theoxidizing agent), to limit the duration at which the microorganism isexposed to the agent, or to use an agent with lower toxicity, evenshould that agent have less desirable redox properties. In addition,bacteria that have been exposed to a toxic redox agent can be treatedafter exposure to a counteracting agent. For example, should the toxicredox agent be an oxidizing agent, the addition of a reducing agent suchas beta-mercaptoethanol or dithiothreitol can reduce the effects of theoxidizing agent.

It should be noted that one of the goals of the use of the redox agentsis to allow electrophoresis to occur at a lower potential, both so as tominimize the production of harmful redox products (e.g. chlorineproducts from chloride), and so that optical detection can occur usingITO electrodes, which can be harmed by high potentials. Thus, the cellpotential of the redox pairs chosen for the application is preferablyunder 2 V (the potential at which ITO begins to be affected), and evenmore preferably under 1 V and most preferably under 500 mV, since therange of potentials between the lowest potential at whichelectrophoresis occurs (i.e. 500 mV) and the endpoint (i.e. 2 V) willgive some measure of control over the rates of electrophoresis. Even inthose cases where the standard cell potentials of the redox agents maybe outside of these ranges, the use of differing concentrations ofoxidizing agent and reducing agent can provide a cell potential thatallows for useful operation.

Passivation

Redox products generated at the anode and cathode can be potentiallyharmful to the molecules and materials being transported to thesesurfaces. For example, many of the redox reactions generate H⁺ ions atthe anode, which cause a local reduction of pH. This reduction in pH, iflarge enough, can disrupt nucleic acid hybridization, denature proteins,interrupt protein-protein or protein-nucleic acid interactions, or killbacteria. Other redox products that are of potential danger also includestrong bases, and strong oxidizing or reducing agents. In order toprevent these products from interfering with the molecules or materialsto be detected at the anode or cathode, it is preferable to have apassivation layer over the electrode.

In general, it is convenient for this passivation layer to be such thatproteins and nucleic acids are not detrimentally affected by thechemical or physical properties of the passivation layer directly andthat the passivation layer does not have a significantly detrimentaleffect on the redox reactions that occur at the electrode. It ispreferable that the passivation layer be at least 2 nanometers thick,and more preferable that the passivation layer be at least 5 nanometersthick, and most preferable that the passivation layer be at least 25nanometers thick, so that the interaction of the targets 114 and probes116 with the products of redox reactions at the electrodes be reduced.Convenient forms of passivation layers include polymers comprisingeither with polyacrylamide (e.g. Codelink by Amersham) or polyethyleneglycol constituents (e.g. OptiChem by Accelr8), modified with functionalgroups to which probes for detection can be attached.

Inhomogeneity Artifacts

It has been observed that under conditions of 10 mM benzoquinone and 10mM hydroquinone, an indium tin oxide (ITO) electrode separation of 300microns, and a potential of greater than 1.5 Volts and less than thebreakdown voltage of the ITO, an inhomogeneity develops either withsoluble (e.g. nucleic acid coupled with a fluorescent dye) or insoluble(e.g. polystyrene spheres) markers. The inhomogeneity is evidenced byareas of concentration and rarefaction, where the areas of concentrationstart as roughly circular spots hundreds of microns across that elongateand condense into a pattern of cells, in which the borders are areas ofconcentration, and the central regions of the cells are areas ofrarefaction. FIG. 15 is a top-view schematic of an approximately 1 cmdiameter region in which such cells have formed. In general, thisinhomogeneity can be an impediment to the use of accelerated transportvia electrophoresis.

There are a number of methods of reducing this inhomogeneity. In a firstreduction method, the strength of the electrophoretic force can bereduced, either by decreasing the voltage, or by increasing theconductivity of the solution. For example, in a solution of 10 mMbenzoquinone and 10 mM hydroquinone and very low conductivity (e.g. <100μS/cm), the cells do not appear very strongly below 1.4 volts. In asecond reduction method, periods of strong electrophoretic force can beinterspersed with periods of lesser or no electrophoretic force, whereinthe amount of lesser electrophoretic force is preferably less than 50%of the maximal force, and more preferably less than 25% of the maximalforce, and is most preferably less than 10% of the maximal force. Ingeneral, the period of strong electrophoretic force should be less thanthat at which the cells first form, and such periods are preferably nomore than 5 seconds, and more preferably no more than 2 seconds, andmost preferably no more than 1 second. The periods withoutelectrophoretic force are conveniently substantial enough to allowdiffusion of ions to distances that are large compared with the verticalsize of the cells (i.e. the distance between the electrodes), and arepreferably more than 100 milliseconds, and more preferably more than 300milliseconds, and most preferably more than 1 second. In a thirdreduction method, it is convenient to allow liquid flow to break up thecells, such as through the use of temperature convection aided byunequal heating of the walls of the chamber 805, or through movement offluid through the chamber 805.

It should be noted that while ITO or other transparent electrodematerial is preferable for real-time monitoring via visible indicators,this does not mean that both the cathode and the anode need to becomprised of ITO. In other instances, it can be preferable for one ofthe electrodes to be transparent, allowing observation into the reactioncell, while the other electrode to be a relatively non-reactive, opaqueelectrode, such as gold or a refractory metal, such as platinum,palladium, or iridium which are stable in electrophoresis. In thesecases, the resistance in the metallic electrode will be very small,which can reduce the inhomogeneity effects above, and furthermore, thepotential on the metallic electrode may not have the same deleteriouseffect as on the ITO electrode (e.g. with a Pt electrode), allowinghigher potential to be used in the cell.

Alternatively, both electrodes can be opaque, with one electrode beingcoated with gold. In this case, the detection can be made optically viasurface plasmon resonance.

Combination of Mixing and Electrophoretic Reaction Acceleration

Given that the electrode 200 is small relative to the lateral dimensionsof the cell, application of force towards the electrode 200 will resultin relatively even distribution of the tagged target 275 on theelectrode. If the specific location 170 is small relative to the size ofthe electrode 200, this will result in only a small fraction of thetagged target 275 being bound to the probe 116. It is thereforeadvantageous to combine the step of mixing with or interspersed with theapplication of the forces towards the electrode 200. This is depicted inFIG. 16A, a schematic block diagram of a reaction involving bothvertical forces and horizontal forces so as to accelerate the reactionof a tagged target 275 with the probe 116. The methods of providingmixing, such as horizontal forces, will be discussed in greater detailbelow, but can be considered to include physical mixing of the medium inthe cell (e.g. through the use of a physical stirring mechanism, pumps,electroosmotic flow, surface wave acoustics, and other means), the useof horizontal electrophoretic forces on the targets 114, the use ofmagnetic forces on the targets 114, and other convenient means. Thoseforces comprising bulk flow of the solution (e.g. electroosmosis,stirring, pumps, and surface wave acoustics) are particularly easy toimplement. The vertical forces can comprise electrophoresis,dielectrophoresis, filtration, magnetic field attraction and other suchforces as will bring the tagged target 275 (or a suitable target 114that is not tagged) into proximity with the probe 116.

It should be noted that the use of “vertical” and “horizontal” is usedin relation to the surface of the electrodes, and is not related togravity, up/down or other coordinate schemes. Given the orientation ofthe diagrams, horizontal can be understood in this context to beparallel to the electrode (or more generally, the surface on which theprobe resides), while vertical can be understood in this context to beperpendicular to the electrode.

For example purposes, the target 114 is a single stranded DNA 470, andthe tagged target 275 additionally comprises an electrophoretic tag 270.The probe 116 comprises a complementary single stranded DNA probe 480,which is attached to the substrate 120. Vertical forces will tend tomove the tagged target 275 vertically towards the probe 480, whereas thehorizontal forces will allow the tagged target 275 to interact withprobe 480 at various locations 170 within the array 190.

FIG. 16B is a graph of the electrical potential causing movement of thetagged target 275 vertically, in time relation to the horizontal forcescausing mixing of the tagged target 275. For purposes of this graph, apositive horizontal force is considered to be in a constant arbitrarydirection along the substrate 120. Furthermore, a positive verticalforce is considered to be in a direction that encourages the movement ofthe tagged target 275 towards the probe 480. As can be seen from thefigure, the horizontal force is relatively constant. However, thevertical force varies in time, and is sometimes approximately neutraland at other times very strong. The vertical force is releasedperiodically in order to allow the tagged target 275, which can becomeenmeshed on the substrate 120 during the application of the verticalforce, to move laterally. The vertical force is applied initially for along duration T7 in order to bring the tagged target 275 near to theprobe 480. Once the target is in close proximity to the surface of thesubstrate 120, subsequent applications of vertical force can be eitherof shorter duration, or of lower magnitude, or both.

Consider, for example, a horizontal force that is sufficient inmagnitude and in duration such that during the course of the reaction,the tagged target 275 moves approximately the width of a location 170.In such case, the location 170 will encounter approximately twice thetagged target 275 than it would without the application of horizontalforces, assuming minimal diffusion.

It is also within the spirit of the present invention for the horizontalforces to switch direction, so that the target 275 moves back and forthover the probe 116. In such case, the target 275 will have multiplepossibilities of interacting with the probe, and will thereby increaseits binding. Also, if the probe is attached through a hydrogel coating,some probe 116 may be sterically hindered from interacting with thetarget 275 if the target is moving from one or another direction, and itcan be advantageous for the target 275 to move back and forth across theprobe so as to provide different movements of the target 275. Also, inorder to increase the amount of binding, the rate of horizontal movementcan be decreased, or the rate of vertical movement increased.

Control of Mixed Vertical/Horizontal Reaction Forces

FIG. 17A is a schematic block diagram of the means of controlling thehorizontal and vertical forces. The electrode 200 lies on the substrate120 and is surrounded by a horizontal force applicator 520. Differentmeans of applying horizontal force will be described in detail below.The applicator 520 is connected to a controller 510, which in turnreceives input from a detector 500. The controller controls both themagnitude of horizontal force applied by the applicator 520, as well isthe vertical force that is directed by the electrodes 195 and 200. Thedetector 500 monitors tagged target 275 that is in close proximity tothe electrode 200 in real time. That is, tagged target 275 that iswithin tens or hundreds of nanometers of the electrode 200 is detected,whereas other tagged target 275 at a further distance from theelectrode, is not. The means by which this real-time monitoring isperformed by the detector 500 will be discussed in greater detail below.

FIG. 17B is a schematic block flow diagram of the operation of thesystem of FIG. 17A. In the step 530, the controller 510 causes avertical force to be exerted between the electrodes 195 and 200 suchthat the tagged target 275 moves towards the electrode 200. In a step532, input from the detector 500 is used by the controller 510 todetermine whether an increasing amount of tagged target 275 is beingdetected that is juxtaposed to the electrode 200. If increasing targetis being detected, continued vertical forces are applied in the step530. If no new tagged target 275 is detected by the detector 500, thecontroller relaxes the vertical force in a step 534. In a step 536, thehorizontal force or flow is either maintained or activated at thispoint. Because of the relaxation of vertical force in the step 534,tagged target 275 diffusing from the surface of the electrode 200 comesunder the influence of the horizontal force or flow and moves laterallyalong the surface of the electrode 200. In a step 538, the detector 500monitors the amount of tagged target 275 juxtaposed to the surface ofthe electrode 200. If the amount of target is still decreasing, therelaxation of the vertical force in the step 534 is maintained.Alternatively, a fixed amount of time can be allowed to elapse. Once theamount of target detected by the detector 500 is relatively steady, orthe fixed amount of time elapses, the cycle is repeated beginning withthe step 530.

Horizontal Forces and Flows

There are a number of different horizontal forces and flows that may beused within the spirit of the present invention. Among these includeelectrophoretic forces, electroosmosis, acoustic waves, mechanicalstirring, and fluid pumping. For example, in FIG. 4B, lateral electrodes210 and 220 can be used to apply horizontal forces to tagged targets275. In such case, the magnitude of the vertical electric field can beadjusted by the potential on the reference electrodes 195, in relationto the magnitude of the horizontal electric field from the electrodes210 and 220.

With respect to acoustic waves, piezoelectric actuators can be placedeither on the substrate 120 or on the cover 111 in a topologicalarrangement such that under a high frequency control signal, surfaceacoustic waves in the glass cause mass transport of the fluid in whichthe tagged target 275 is suspended. In such case, a convection currentis created within the cell which maintains a constant laminar flowacross the surface of the substrate 120. By alternating the control ofthe piezoelectric signals, periods of turbulent mixing can be alternatedwith periods of laminar flow.

Mechanical or electroosmotic pumping can also be used to create laminarflow across the surface 120. While mechanical pumping is convenient forlarger volumes, electroosmotic pumping can be used to assist even in thecase of extremely small volumes. In such case, the electroosmoticsurfaces can be incorporated either into the substrate 120, or moreconveniently into the cover 111, since the substrate 120 is oftencovered by a custom surface used primarily to bind probe 116 and toreduce the amount of nonspecific binding, and which may be a lesseffective surface for creating electroosmotic forces.

FIG. 18A is a perspective diagram of a mechanical stirring system thatcan be used within a microtiter plate well 550. The microtiter platewell 550 has a round probe electrode 560 on its bottom surface connectedto the outside of the microtiter well 550 by an electrical trace 558. Areference electrode 570 is immersed within the analyte fluid whoseheight is represented by the dashed line 556. The reference electrode570 is mounted on a shaft 552 which has both mechanical and electricalconnections to actuators not shown in the figure.

During operation, the shaft 552 provides not only electrical connectionsthrough which a potential bias can be placed on the reference electrode570, but in addition, the shaft 552 causes the reference electrode 570to rotate. Because of the viscosity of the analyte fluid, the fluidconvects in a circular motion around the microtiter plate well 550, withroughly equal degrees of movement within each radius from the center ofthe well 550. By reversing the direction of rotation of the shaft 552 inthe reference electrode 570, turbulent flow within the well 550 can beinduced.

It should be noted that due to the symmetry of the situation, and due tothe desire to have roughly equal amounts of conductive flow for each ofthe probe locations 170, it can be preferable for the probe electrode560 to have circular symmetry. FIG. 18B is a top-view diagram of theprobe electrode 560. The electrode 560 is arranged as an annular ring ofconductive material attached to the trace 558. Probe locations 170 arearranged around the ring, and are roughly equidistant from the center ofthe microtiter well 550. In this arrangement, there is no preference inthe electric field or the association of targets 114 to probes 116 basedon physical location. Furthermore, conductive laminar flow induced bythe electrode 570 will cause tagged targets 275 to move in a circularmovement around the electrode 560.

It can alternatively be convenient for the reference electrode 570 notto be symmetrically placed at the bottom of the shaft 552, but rather tobe asymmetrically disposed. In such case, the electric field directionwill rotate with the shaft 552, providing the benefits of changingelectric field directions, as described above.

The microtiter plate assays can be run either one at a time, or multipleassays at a time. FIG. 19A is a perspective diagram of a microtiterplate 590 with a set of electrodes 570 and shafts 552. The electrodes570 and shafts 552 each fit into single microtiter wells 550 arranged ina grid. The electrodes 570 and shafts 552 can either rotate or be in afixed position.

Depending on the arrangement, it is convenient either to have all of theelectrodes 570 and shafts 552 be fixed with respect to each other,allowing for parallel operation in all wells and for simple andinexpensive construction, or individual electrodes 570 and shafts 552can be independently controlled. Alternatively, instead of atwo-dimensional array of shafts 552 and electrodes 570 as shown, therecan be a one-dimensional array, in which a single row of wells on themicrotiter plate are processed at one time.

The microtiter plate 590 can be of unitary construction, oralternatively be constructed of a top plate and a bottom plate, in whichthe top plate is made of plastic and defines the sides of the wells,whereas the bottom plate is made of plastic, glass or other substratethat is substantially flat, and which is coated with a material reducingnonspecific binding and to which probes 116 can bind. In such case, thebottom plate is adhered to the top plate using adhesive, preferablyafter the printing of the array 180 of probe locations 170. For purposesof the present invention, it is convenient for electrodes to be placedon the bottom plate prior to the printing of the probes 116 or theadhering of the bottom plate to the top plate.

FIG. 20A is a top view of the arrangement of well electrodes 598 on abottom plate 592. The well electrodes 598 can be square (as shown),rectangular, ellipsoidal, circular or annular (as in electrode 560), andare connected to end pads 594 via traces 558. These traces can be ofrelatively constant width, but are preferably narrower at the locationsof the wells (denoted by dotted lines), where the majority of theelectrically conductive area is preferably that of the well electrodes598. It is also within the spirit of the present invention for theelectrically-conductive traces that are not part of the electrode 598 tobe covered with a non-conductive coating (e.g. semiconductor materials,ceramics, oxides, and other materials), but this is an additional stepand cost of manufacture and may not be always convenient. In addition,there may be multiple traces per well, such as would be convenient withelectrodes not underlying probe locations.

There is an attachment pad 594 for each electrode 598, to which theelectrical attachment is made. This is less convenient when the numberof wells 550 (and therefore electrodes 598) becomes very large.Alternatively, multiple electrodes 598 can be connected to a single pad593, as shown in FIG. 20B, a top view of the arrangement ofelectrically-connected well electrodes 598 on a bottom plate 592. Inthis case, there is a single electrode 593 to which all electrodes 598are electrically connected. Even if not all electrodes 598 are insimultaneous use, this arrangement allows for simple electricalconnectivity, and no harm occurs with the parallel connection with theunused electrodes 598. Other arrangements are also within the spirit ofthe present invention, such as connection of all electrodes 598 within asingle row or column of the array of wells 550, whereas each row orcolumn is connected to a different attachment pad 594.

As described above, the bottom plate 592 is adhered to a top plate. Ifthe bottom plate 592 is smaller than the top plate, the pads 594 or 593can be grabbed by an electrical attachment device from underneath theplate (access through the top and sides is prevented by the top plate).An alternative arrangement that succeeds regardless of the relativesizes of the top plate and the bottom plate 592 is shown in FIG. 19B, aperspective view of a top plate 591 comprising access ports 597. In thisarrangement, the access ports 597 provide side access to connect withthe pads 593 or 594. The access ports 597 are placed according to thelocations, number and sizes of the pads, and access to multiple padsfrom a single port 597 is within the spirit of the present invention.

An alternative arrangement is for the bottom plate 592 to be uniformlyconductive, and maintained at a ground potential. In such case, theelectric field within each microtiter well 550 can be independentlyadjusted by adjusting the potential on the corresponding electrode 570.In the case where one electrode 570 is operating at the time, the use ofthe uniformly conductive bottom plate 592 is straightforward. Whenmultiple wells 550 are simultaneously being operated via a multiplicityof operating electrodes 570, it is optimal if the electricalconductivity of the analyte solution in each well is low relative tothat of the bottom plate 592. The electrical conductivity of the analytesolution can be adjusted by, for example, lowering the concentration ofions in solution.

Microtiter wells can be used within the present invention without use ofpermanent electrodes on the bottom plates 591. FIG. 21 is a perspectiveside diagram of an integrated electrode 600 for microtiter plates. Theintegrated electrode 600 comprises three sets of independentlymodulatable electrodes: a reference electrode 606, a first lateralelectrode 610 and a second lateral electrode 612. Each of theseelectrodes can, in turn, comprise electrodes that can be independentlycontrolled.

The electrodes 606, 610 and 612 are mounted on a shaft comprisingvertical members 608 and plate 602, which provide both physical supportas well as electrical connections. Input electrical control is providedthrough shafts 604, which comprise both physical and electricalconnections as well. The number of shafts 604 can be as small as one.The lateral electrodes 610 and 612 correspond roughly to electrodes 210and 220 of FIG. 4B, and the reference electrode corresponded roughly tothe electrodes 190. These electrodes are conveniently comprised of arelatively unreactive metal with high conductivity, such as gold. It ispreferable for the lateral electrodes 610 and 612 to be relatively thin,and can also taper at their interior edges to maintain a flat lowersurface, allowing electric fields to be controlled near to the bottom ofthe electrode 600.

The integrated electrode 600 is placed in a microtiter plate well 550with the bottom surface of the lateral electrodes 610 and 612 placed onto or very near to the bottom of the well 550, with the array 180 ofprobes 116 sitting between the two lateral electrodes. The electrode 600performs similarly to the arrangement of FIG. 4B. At the conclusion ofeach assay, the electrode 600 is removed from the well 550 and washedwith strong applied electrical potentials, physical agitation in asolution, and possibly chemical washes in strong acids, oxidizingreagents and other cleaning solutions. It is also within the spirit ofthe current invention for the electrode 600 to be turned in a roughlycircular or in a back and forth motion so as to mix and/or move thetarget 114 in accordance with the methods described above (e.g. seeFIGS. 33A and 33B).

Acceleration of Signal Generation Using Electrophoretic Manipulation

In some cases, the tagged target requires subsequent exposure tosubstrate in order to generate signal that can be detected by a varietyof means. For example, chemiluminescence requires the addition ofsubstrate to enzyme tag in order to generate chemiluminescent signal.Electrophoretic forces can be used to drive enzyme reaction by bringingsubstrate in close proximity to enzyme and then, upon enzymaticconversion of substrate to opposite electric charged state, can be usedto drive converted substrate away from enzyme, rapidly enabling morerapid conversion of the substrate by the enzyme.

Washing-Detection

Overview

In the sections above, numerous references are made to discriminatingspecifically bound versus nonspecifically bound material by increasingthe bias on an electrode that pulls the electrophoretic tag 270 and theattached target 114 away from the probe 116 with increasing amounts ofelectrostatic force. This process is described in more detail in FIG.22, a schematic block flow diagram of discrimination usingelectrophoretic force.

In a step 400, optional chemical washes are used to remove loosely-boundnonspecifically bound material. These chemical watches can includelow-salt, high pH, low pH, or other chemical treatments which lower thebinding force between the target 114 and the probe 116.

In a step 402, the chemical washes performed in the step 400 arereplaced with a stability buffer that tends to increase the bindingforce between the target 114 and the probe 116. The stability bufferreduces the chances that the target 114 and the probe 116 will separateadventitiously. It should be noted that in the absence of the step 400,the step 402 can also optionally be eliminated.

In a step 404, the tagged target 275 attached to the probe 116 isvisually monitored. In general, this will involve either capturing animage of the array 180, or scanning the array 180 in a manner to bedescribed below. In general, the detection means is matched to the typeof indicator component used in the electrophoretic tag 270. It isimportant that the tagged target 275 which is not associated with theprobe 116 is not monitored in this situation. Methods of real-timedetection for discriminating bound from unbound electrophoretic tag 270are described above and below. In a step 412, the visual data that iscaptured is stored.

In a step 406, the net vertical bias away from the probe 116 isincreased in a manner to be described below. This increase willgenerally be incremental in a manner shown in FIG. 9. In a step 408, itis determined whether or not the maximum stringency from theelectrophoretic force has been reached. If it has not been reached, newvisual data is captured in the step 404. If the maximum stringency hasbeen reached, the differences in binding for each electrophoreticstringency is computed from the differences between successive capturedvisual data in a step 410, as described below.

It should be noted that for visual detection, there are a variety ofdifferent illumination schemes that can be employed. Some of theseillumination schemes require specialized condensers for use in phase andother types of microscopy. For use in the detection of scattered light,as well as with the use of fluorescent, quantum dot and upconvertingphosphors, and certain other modes of detection, the use of other formsof illumination can be used. In many cases, the use of evanescent waveillumination can be of particular use, because the light that does notinteract with the target 114 or its tag 270 can be oftentimes preventedfrom interfering in the detection, and because the only tags 270 thatwill interact with the light will be those tags that are proximal to theprobes 116 on the surface or the substrate. The following discussionwill go into detail into the means by which evanescent illumination canbe used in visual detection of the tagged target.

Evanescent Illumination Detection Using Parallel Beam Illumination

FIG. 23A is a cross-sectional schematic of an embodiment of the presentinvention in which a prism 1140 on the top surface is used to introducelight into the slide waveguide 1120. The prism 1140 shown in the figureis a triangular parallelopiped, in which one surface is placed on thetop surface 1122 of the slide 1120, and the acceptance surface 1142faces roughly in the same direction as an edge 1123 of the slide 1120.Roughly parallel light rays 1132, which are preferably nearlyperpendicular to the surface 1142 but which can be non-normal andtherefore refracted at the surface 1142, enter the surface 1142 withlittle reflection. These light rays 1132 encounter the bottom surface ofthe prism 1140, and due to the flatness and juxtaposition of the bottomsurface of the prism and the top surface 1122 of the slide, the lightrays 1132 bridge the gap between the prism 1140 and the slide 1120,entering the slide 1120. The direction of the light rays 1132 is chosenso that the rays 1132, when encountering the bottom surface 1124 of theslide 1120, will nearly all reflect off of the surface 1124, impingingat greater than the critical angle between the surface 1124 and themedium (generally air) below.

The top surface 1143 of the prism 1140 is chosen so that all of thelight rays 1132 that enter the prism 1140 are captured into the slide1120, and it is of some convenience that the angle between theacceptance surface 1142 and the top surface 1143 of the prism 1140should be roughly perpendicular. It should be noted, however, that ifthe apex 1145 of the prism were to be extended far enough along theslide, that ray paths reflected off of the bottom surface 1124, movingupwards to the top surface 1122, could encounter the bottom surface ofthe prism 1140, resulting in “escape” of the light from the slide. Thisshould be avoided by not extending the apex 1145 too far distally alongthe slide 1120.

While the parallel rays 1132 are shown to be nearly perpendicular to theacceptance surface 1142, and therefore exhibit almost no refraction, itis within the spirit of the present invention for the light rays 1132 toenter non-perpendicularly to the surface 1142, such that the refractedray paths have an appropriate trajectory, resulting in nearly totalinternal reflection within the slide.

FIG. 23B is a cross-sectional schematic of a prism 1140 on the topsurface of a slide, in which light is internally reflected within theprism prior to introduction of the light into the slide 1120. In manycases, it is preferential to keep device components roughlyperpendicular to one another in order to aid alignment, and in thiscase, the incident light rays 1132 can be nearly perpendicular to theedge 1123 of the slide 1120 (and therefore parallel to the top surface1122 of the slide 1120). The acceptance surface 1142 can be parallel tothe slide edge 1123, so that the rays 1132 are perpendicular to thesurface 1142, thereby limiting reflection at the surface 1142.

After an internal reflection on the top surface 1143 of the prism 1140,the light rays 1132 now have the proper angle into the slide 1120 so asto exhibit total internal reflection. It should be noted that the angleof the ray paths 1132 after reflection on the surface 1143 of the prism1140 will be twice that of the slope of the prism 1140—therefore, theslope of the top surface 1143 needs to be reasonably small in order tomaintain total internal reflection of the ray paths 1132 within theslide 1120.

FIG. 24A is a cross-section schematic of the prism arrangement of FIG.23, extended so that the disposition of the distal parallel ray paths1132 can be seen. Parallel ray paths 1132 enter the prism 1140, and thenenter the slide 1120. Because of the parallel nature of the ray paths1132, the pattern of reflections within the parallel walls of the slide1120, acting as a waveguide, are maintained along the length of theslide 1120. If the rays are bounded by the parallel topmost ray path1133 and the bottommost ray path 1135, and the top surface 1122 andbottom surface 1124 are parallel, illuminated sections 1146 will berepeatedly interspersed with unilluminated sections 1148 along thelength of the slide 1120. This will cause significant differences inreporter 1110 illumination along the slide 1120. This non-uniformity canbe to some extent handled by the use of a wide beam of illumination, butit will generally be difficult to modulate beam width so thatillumination is precisely uniform.

Evanescent Illumination Detection Using Convergent Beam Illumination

FIG. 24B is the cross-sectional schematic of FIG. 24A, modified by theuse of convergent illumination instead of collimated illumination.Converging illumination 1131 enters the prism 1140, during which it isrefracted somewhat at the acceptance surface 1142. It is convenient thatthe point of convergence of the ray paths not be at the interfacebetween the prism 1140 and the slide 1120, since any imperfections inthe glass or contaminants (e.g. dust) at the interface could contributeto light scattering. Scattered light would not necessarily maintaintotal internal reflection in the slide 1120, and so the point ofconvergence is preferably either before or after the point at which thelight 1131 enters the slide 1120.

Looking at the light in the slide, the trajectories of the topmost raypath 1133 and ray path 1135 can be observed. As can be seen, there is norepeating nature to the areas of illumination and non-illumination forthe ray paths 1133 and 1135. Indeed, there is a large range of ray pathangles within the light 1131, so that indeed much of the top surface1122 of the slide 1120 is illuminated after only a small number ofinternal reflections, and given a very large number of reflections, theillumination of the top surface 1122 becomes nearly uniform. As before,a wider beam will generally result in somewhat more uniform illuminationin the case of fewer reflections.

It should be noted that a divergent spread of illumination entering theacceptance surface 1142 would have a similar effect to a convergentillumination, resulting in nearly homogeneous evanescent illumination ofthe top surface 1122.

Evanescent Illumination Detection Using Non-Uniform Illumination

While the embodiments of FIGS. 23A and B and FIGS. 24A and B can be usedwith the prism 1140 and/or associated illumination source being in afixed location, possibly near the end of the slide, it is also in thespirit of the present invention that the prism 1140 and/or itsassociated illumination source can move to illuminate different areas ofthe slide, particularly wherein the illumination is intentionallynon-uniform.

FIG. 24C is a schematic cross-sectional diagram of a slide illuminatorin which the slide is non-uniformly illuminated. The prism 1140 sits onthe top surface 1122 of the slide 1120, and accepts parallel rays 1132from a collimator 1170. A fiber optic cable 1174 conveys light to thecollimator 1170, and light rays diverging from the end of the fiberoptic cable 1174 are captured by and converged by a lens 1172, producingcollimated rays 1132.

As in FIG. 24A, the light rays 1132 enter into the prism 1140, andthence into the slide 1120, wherein they then reflect multiply againstthe top layer 1122 and the bottom layer 1124, illuminating the topsurface 1122 at regular intervals 1. The length 1 can be computed to be2*d/tan θ, where d is the thickness of the slide and θ is the anglecomplement of the angle of incidence of the light onto the top or bottomsurface of the slide.

A detector 1160 is positioned over the spot of illumination 1146 on thetop surface 1122 of the slide 1120, and detects a signal resulting fromthe evanescent illumination of the reporters 1110 residing on the topsurface 1122. It should be noted that the detector 1160 could also bepositioned over integral multiples of 1 in distance on the top surface,which is of special convenience should there be topological constraintson the location of the detector 1160 relative, for example, to the prism1140. It should be understood that the detector technology can compriseboth imaging devices (e.g. CCD or CMOS cameras operating with arelatively constant light source) and non-imaging devices (e.g. aphotomultiplier tube (PMT) operating in conjunction with a laser scannerilluminating the surface through prism or other coupling).

While this arrangement is effective for illuminating material at aparticular position relative to the prism 1140, this arrangement canalso be used to illuminate many areas on the top of the slide 1120. Thiscan be accomplished, for example, by sliding the prism 1140 and theassociated collimator 1170 in concert over the top surface of the slide1120. A movement of the prism 1140 and collimator 1170 would result in aconcomitant movement of the spot of illumination 1146 of an equalamount.

Alternatively, the prism can be kept in a single location, and thecollimator 1170 can be translated horizontally or vertically,maintaining its orientation, such that the point of entrance of thelight ray 1132 into the prism is altered. This will translate the lightray 1132 laterally within the slide. Furthermore, rotation of thecollimator 1170 would have a translational effect on the position of thespot of illumination 1146. It is also within the spirit of the presentinvention for there to be a combination of more than one of themovements of the collimator 1170, possibly in concert with movement ofthe prism 1140, in order to effect translation of the spot 1146 alongthe top surface 1122.

Evanescent Illumination Detection Using Top Surface Thin Film Waveguide

Another embodiment of the present invention is to make a very thinwaveguide, rather than using the slide, which generally has a thicknessof a millimeter or larger. This can be accomplished in a variety ofways. For example, the slide itself can be constructed as a film,possibly of a flexible high index plastic material. This may not beconvenient in certain applications, including such cases where the filmis to maintain structural rigidity; the plastic material isinappropriate for the biological and chemical reactions used in thedetection process, and allowing the material to bend will potentiallyallow light to escape when internal reflection angles become less thanthe critical angle.

An alternative embodiment is shown in FIG. 25A, a schematiccross-section of a high index thin film waveguide 1180 deposited on aslide substrate by physical vapor deposition (e.g. sputtering orevaporation), by chemical vapor deposition, by spin coating, dipcoating, or by other means that provides a film of roughly uniformthickness. Furthermore, graded index of refraction thin films can begenerated using sol-gel and ion exchange methods. A review of themethods for producing such thin waveguides is provided in “Planarintegrated optical methods for examining thin films and their surfaceadlayers” by Plowman, Saavedra and Reichert (Biomaterials (1998) 19, pg.341-355).

The thin film waveguide 1180 is comprised of a material that has asubstantially higher index of refraction than the underlying slide 1120.The material is conveniently Ta₂O₅, which is commonly used in thehigh-index layers in the production of thin-film interference filters,although other materials can be used, such as TiO₂, silicon nitride,ion-doped silica, and ion-doped glasses. The thickness of the waveguideis generally on the order of a wavelength of the guided light, which inthis case will typically be in the visible or ultraviolet (UV) range,and can conveniently be on the order of 100-5000 nm, and is morepreferably 150-2000 nm. Because of the small thickness, only one or afew modes are transmitted in the waveguide (i.e. single-mode) as opposedto the multi-modal transmission of light in a thick waveguide (e.g. theslide).

Coupling of the incident illumination into the thin film waveguide canbe accomplished in a number of ways. FIG. 25A is a schematiccross-section of an end-illuminated thinfilm waveguide 1180 integratedwith a slide 1120. A fiber optic cable 1174 transmits light along asingle-mode fiber 1175, which terminates in a coupler 1182. The coupler1182 can also be seen in FIG. 25B, a schematic top view of the coupler1182 and the slide 1120 of FIG. 25A. Light exiting the fiber 1174encounters a conditioning lens 1177 that is used to adjust thedivergence of the emergent light rays, and may be either convergent ordivergent. The light is then passed through a cylindrical lens 1178 toconverge the beam in a single dimension, oriented in such a way that theemerging light lines up roughly with the waveguide 1180. Optimally, thefocal point is roughly coincident with the edge surface of the waveguide1180. The beam so constrained gains significant admittance into thewaveguide 1180.

The coupler 1182 encapsulates the terminus of the fiber optic cable1174, as well as the conditioning lens 1177 and cylindrical lens 1178. Apositioning lip 1183 on the top front of the coupler 1182 is used toposition the coupler 1182 onto the slide 1120 with the optics arrangedto couple light into the waveguide 1180.

It should be noted that the optical arrangement of lenses can be variedwithin the teachings of the present invention. For example, the fiberoptic cable can be butt-end juxtaposed directly to the edge of thewaveguide 1180. Alternatively, the conditioning lens 1177 can be leftout, in part depending on the placement of the cable 1174. Also, thecylindrical lens 1178 can be omitted, given a conditioning lens 1177that converges on the edge of the waveguide.

It should be noted that there can be some leakage of the beam eitherabove the waveguide 1180 or into the slide 1120, which for very narrowwaveguides can comprise the majority of the light from the cylindricallens 1178, since coupling tends to be inefficient. With leakage abovethe waveguide, the coupler 1182 has an overhang that lies on top of thewaveguide 1180, both helping in aligning the coupler 1182 so that lightfrom the fiber optic cable 1174 enters the waveguide 1180, and blockinglight escaping from the coupler 1182 forwards. With leakage into theslide 1120, small amounts of light that leak into the slide 1120 willtend to be constrained within the slide 1120 (acting as a waveguide).Other light with a higher angle (so that it doesn't reflect) will firstencounter the bottom surface of the slide 1120, where it will escape andalso not affect the evanescent or other illumination above the waveguide1180.

An alternative method of coupling the illumination into the thin filmwaveguide 1180 is to place a grating onto the surface of the waveguide1180. The principle of operation and construction of such a gratingcoupler is provided in Plowman, et al. (reference provided above). FIG.25C is a schematic cross-section of a grating 1181 on the surface of thewaveguide 1180, with incident illumination thereby captured into thewaveguide 1180. The grating is positioned on the top surface of thethinfilm waveguide 1180, with input light 1202 directed from below ontothe waveguide 1180. The grating can also be positioned at thewaveguide/substrate interface, or at any interface in a multi-layerwaveguide 1180. Furthermore, the waveguide 1180 can be illuminated fromabove as well as below.

A prism can also be used to couple light into a thin film waveguide1180. FIG. 25D is a schematic cross-section of a thin film waveguide1180 wherein light is coupled to the waveguide 1180 via a high-indexmaterial prism 1200. It should be noted that the input light 1202 entersthe waveguide 1180 close to the edge of the prism 1200, since high indexof refraction prism 1200 material that overlies the waveguide 1180beyond the point of coupling will permit light in the waveguide 1180 toescape. The input light 1202 can, therefore, either be a narrow,collimated beam that is directed at the vertex 1204 of the prism 1200,or can be a beam of light that converges near the vertex 1204 (e.g. viaa spherical convex lens or plano-convex cylindrical lens).

It should be noted that the input light 1202 need not be roughlyperpendicular to the face of the prism 1200, and it can refract at thatsurface so that it is at the proper incident angle into the waveguide1180 at the proper location. It should also be understood that it iswithin the teachings of the invention that the prism 1200 for waveguide1180 coupling to have a triangular, trapezoidal, or other cross-section.

Evanescent Illumination Detection Using Single Bounce Non-WaveguideArchitectures

In the embodiments above, illumination that is captured into thewaveguide 1180 is introduced in the direction of the top surface of theslide 1120, from which the detection is performed. It should also benoted that evanescent waves can be created through systems in which thelight is not captured into a waveguide 1180, but simply reflects onceagainst the top surface of the slide 1120. At the location of thereflection, an evanescent wave is created. It should be noted that thisarchitecture, though organized in a somewhat similar architecture,shares considerable theoretical overlap with the embodiment of integralreflections as in FIG. 24C, except that the light, after illumination ofthe appropriate top surface location, is not constrained with the slide1120.

FIG. 26A is a schematic cross-section of evanescent illumination of aregion without use of a waveguide 1180. A trapezoidal prism 1190 isjuxtaposed to the bottom surface 1124 of the slide 1120. Incoming light1132 enters the prism 1190 on an acceptance surface 1192, andtransverses the prism 1190, encountering the slide on its bottom surface1124. The index of refraction of the prism 1190 and the slide 1120 arechosen to be similar, so that the light enters the slide 1120, generallywith little or no refraction.

The light 1132, refracted at the boundary of the prism 1190 and theslide 1120, traverses the slide 1120 where it encounters the top surface1122 of the slide 1120, and the angle of incidence is chosen to begreater than the critical angle at that surface 1122. Thus, the lightreflects off of the top surface 1122. As shown in FIG. 26A, the light1132 then re-enters the prism 1190 and then exits via emergent surface1194. It should be noted that the goal is to illuminate a region of thetop surface 1122 of the slide 1120, so that the disposition of the lightafter the reflection on the top surface 1122 is of less concern. Thus,instead of a trapezoidal prism 1190, the prism can be truncated suchthat light entering the slide 1120 from the prism 1190 then remains inthe slide 1120, with the slide 1120 then functioning as a waveguide.

Indeed, it can be of some convenience for the prism 1190 to extend onlyto the point where the light 1132 enters the slide 1120, being otherwisetruncated. This arrangement provides more room for a detector to bemounted underneath rather than above the slide, which may be useful incertain applications. Alternatively, given proper stand-off optics,detection can be made through the prism 1190.

Alternatively, the prism 1190 can be constructed so as to allow a windowfor detection. FIG. 26B is a schematic cross-section of evanescentillumination according to FIG. 26A, in which the prism 1190 has a window1193 through which the detector 1160 detects events on the top surface1122 of the slide 1120. The window 1193 is either fabricated duringprism 1190 construction, or is ground into the prism 1190 after theprism 1190 is fabricated. For example, the window 1193 can be producedby a conical grinding wheel, opening a hole directly below the areailluminated on the top surface 1122 of the slide 1120. Within theteachings of the present invention, the window 1193 can be of manytopologies, including conical, rectangular box, trapezoidal trough, orcomplex geometries combining different shapes. Furthermore, instead of awindow 1193, the prism 1190 can be replaced with two “half-prisms” (orentry and exit prisms), each comprising a region that couples with theslide 1120, and the space between the half-prisms comprising a “window”area where a detector 1160 can be placed.

Within this window 1193, the detector 1160 can operate from belowwithout interference from the prism 1190, or can be mounted within theprofile of the prism 1190 should the working distance of the detector1160 be limited.

It should be noted that the indices of refraction in the slide 1120 andthe prism 1190 can differ with the provisos that the angle of light atthe bottom surface 1124 is below the critical angle, allowing the lightto enter the slide 1120, and that the refracted light in the slide 1120encounters the top surface 1122 above the critical angle so that itreflects off of the top surface 1122. Furthermore, however, as the angleof the light at the boundary of the prism 1190 and the slide 1120approaches the critical angle, the partitioning of light betweentransmission and reflection becomes increasingly biased towardsreflection, so that it is preferable for the difference in the indicesof refraction between the prism 1190 and the slide 1120 to be minimized.

In many cases, the area of detection on the slide 1120 is large comparedwith the area illuminated by the illuminator and the area ofillumination on the slide 1120 must be moved relative to the slide.There are three primary means of accomplishing this goal. In the first,the prism 1190 and detector 1160 maintain fixed positions, and the slide1120 moves. In the second, the detector 1160 and the illumination moveindependently—the position of the spot of illumination can be adjustedeither by translating the position of the collimator 1170, or, if thecollimator 1170 is mounted to the prism, by moving the prism 1190. Thearrangement of FIG. 26B has both the collimator 1170 and the detector1160 being placed in fixed position relative to the prism 1190, suchthat movement of the prism 1190 naturally and conveniently repositionsthe illumination and detection means in concert, maintaining a fixedrelationship. It should be noted that mounting of the illumination anddetection means to the prism 1190 does not depend on the presence of thewindow 1193, and can be accomplished conveniently with a trapezoidalprism, such as in FIG. 26A, with the detector 1160 mounted to the flatbottom surface of the prism 1190.

Coupling between the prism 1190 and the slide 1120 can be difficult,given that it is interfered with by dust and other particles, and thetight coupling makes difficult the separation of the two flat interfacesin an operational device. Often, an index matching fluid with an indexof refraction similar to that of the glass of the slide 1120 or theglass of the prism 1190 is used, but this arrangement suffers from dustand other particles that can accumulate within the fluid. Furthermore,excess fluid transferred to the slide (e.g. by smearing or beingexpressed from between the slide 1120 and the prism 1190) canpotentially allow light to leak from the waveguide.

It should be noted that there are three distinct surface areas throughwhich the light interacts with: the surface 1192, the surface 1124, andthe surface 1194. These surfaces can be present on either two differentcomponents (as in FIG. 26A), on three different components (as in FIG.26B), or can alternatively be on a single component, as would be thecase in a molded single piece that could have a cross-sectionsubstantially similar to that of the FIG. 26A or B. All of thesearrangements are within the spirit of the present invention.

An alternative arrangement is presented in FIG. 27A, a schematiccross-section of a prism 1190 that couples light using a flexiblecoupler 1250. The prism 1190 couples the light ray 1132 into the slide1120. To facilitate the coupling, the coupler 1250 is positioned betweenthe prism 1190 and the slide 1120. The coupler 1250 is made of flexibletransparent material and its thickness can range from hundreds ofmicrons generally up to 2 millimeters. The composition of the coupler1250 can include optical curing gels such as NyoGel, flexible opticaladhesives, which can be UV cured, as well as transparent, curablesilicone rubbers. It is preferable that the index of refraction of thiscoupler 1250 should be similar to that of the slide 1120 material or theprism 1190 material, or be of intermediate refractive index. In general,the coupler will be attached to the prism 1190, and can be molded as anadhesive onto the prism 1190.

In order to reduce the potential for air being trapped between thecoupler, it is convenient for the prism 1190 with the attached coupler1250 to be brought onto the slide 1120 at a slight angle, so that airwill be pressed outward from the initial point of contact as fullcontact is made between the coupler 1250 and the slide 1120.Alternatively, the bottom face of the coupler 1250 can be slightlycurved in order to take account of this problem. FIG. 27B is a side viewschematic of a prism 1190 with a curved face coupler 1252. Givencurvature on a bottom face 1254, as shown in the figure, as the prism1190 is lowered onto the slide 1120, air will be forced towards the partof the coupler 1252 for which contact is not yet completed. Thedifference in thickness from one end of the coupler 1252 to the otherend of the coupler 1252 does not need to be large in this instance,though it is preferably greater than 0.5 millimeter.

Other Methods of Illumination and Detection

Other methods of realtime detection are convenient within the spirit ofthe present invention, including confocal microscopy in conjunction withscattering, fluorescence, upconverting phosphors, quantum dots or otherindicators, as well surface plasmon resonance (SPR). Confocal microscopytakes advantage of a very shallow depth of field, such that indicatortags that are drawn away from the probes 116 are out of focus and thelight energy is either dispersed or reduced through spatial filtering.Imaging similar to confocal imaging is also possible using very largenumerical aperture objectives which also have shallow depth of field.Surface plasmon resonance uses an arrangement of components similar tothat of detection using single bounce non-waveguide architectures, asdescribed above, in which the top surface of the glass is coated with areflective, metallic surface which is conveniently gold. In this case,the amount of light reflected by the gold is affected by the presence ofmaterial bound to the probes 116. Surface plasmon resonance is wellsuited to the present invention, in that the gold surface can serve bothas a reflective surface, as well as the electrode for use in reactionacceleration and binding force discrimination.

The methods above have the advantage that targets 114 binding to theprobes 116 are visible and distinguishable even in the presence ofunbound target 114, since only that target that is bound is visible.However, it is further within the spirit of the present invention foralternative arrangements of illuminators and detectors, given thatunbound targets 114 can be removed from the region of the probe 116,either by removal of the solution in which the targets 114 are provided(e.g. as shown below in the case of chambers for the detection ofbacteria), or through the sequestration of the targets 114 in anotherregion. The latter method can involve, for example, the electrophoresisof target 114 to another electrode that is not in the optical patheither of the detector and/or illuminator.

Some of the arrangements that are available within the present inventioncan be understood with reference to two parallel substrates (a lower andan upper substrate) with electrodes on these substrates facing eachother across an internal gap. We can then define from bottom to top fourdifferent surfaces—the lower bottom surface, the upper bottom surface(i.e. with an electrode on which probe is deposited), the lower topsurface (i.e. with an electrode without probe) and an upper top surface.The detector in general will be either below the lower bottom surface orabove the upper top surface (i.e. it is not in the gap between the twosubstrates).

If the detector is below the lower bottom surface, then the electrode onthe upper bottom surface will generally be transparent, except in thecase of surface plasmon resonance. In the case of surface plasmonresonance, the detector must also be below the lower bottom surface. Theillumination can either be below the lower bottom surface, passingthrough the bottom substrate electrode, with back-scattered light,evanescent light (which reflect off of the upper bottom surface), orlight that is meant to excite fluorophores, upconverting phosphors orquantum dots. Alternatively, the illumination can be from within thebottom substrate, as described above. Also, the illumination can be fromwithin the gap between the two substrates, which would generally be bestfor a light scattering application. Alternatively, the illumination canbe from above the upper top surface, transiting through the topsubstrate, through the gap, and then to the upper bottom surface whereit interacts with the target or a tagged target. In those cases, onceagain, the detector can detect either scattered light (e.g. forwardscattered light), or fluorophores, upconverting phosphors, or quantumdots, or the samples can be viewed for brightfield, darkfield, phase orother forms of microscopic imaging (generally using light from acondenser).

If the detector is above the upper top surface, receiving light from thetagged target, in this case the electrode on the upper bottom surfaceneed not be transparent, while the electrode on the lower top surfaceshould be transparent. If the upper bottom surface is opaque, then theillumination must either come from above that electrode surface, or begenerated at the tagged target, as might occur with chemiluminescence.With an opaque upper bottom surface, the illumination can be within thecap (most likely for scattered light analysis), and otherwise mostlikely for scattered light or excitation illumination for fluorophores,upconverting phosphors, or quantum dots. If the electrode on the upperbottom surface is transparent, however, light can be transmitted frombelow, including by evanescent wave illumination as described above.

While the detector is generally an imager (e.g. a CCD or CMOS camera),it can also comprise a laser scanner with a PMT or other light gatheringdevice. In certain cases, the detector can also entail a general lightgathering device (PMT, photodiode, photoresistor) with diffuseillumination. The latter case will be primarily used in those caseswhere averaged signal over an area provides suitable signal, asdiscussed below.

When using a CCD or CMOS camera, the information is obtained pixel bypixel, generally in 8-12 bit grayscale, though in certain cases (e.g.with indicators color-coded for different targets) an RGB image canalternatively be used. In those cases where it is useful or important toregister individual target binding events, there are potentially twomodes of operation. In a first mode, target binding is limited so thatonly a fraction of the pixels register with a signal—most pixels are atsome background level, so that the change from the background level to alevel significantly above background level at a pixel denotes a bindingevent. Depending on the size of the target (and/or its tag), a singlebinding event may correspond to an increase in the signal abovebackground at a number of different contiguous pixels (most imageprocessing software has routines that can group together regions ofcontiguous pixels into discrete “events”). In this case, the dynamicrange of the system ranges from less than 100 targets and as small as 1target (and is limited by the statistical variation of the small numberof targets), to as roughly as high as the number of pixels in the cameradivided by the average number of pixels per target (with a floor ofone), and then divided by a factor approximating 10, which is the“saturation point” at which new targets would more likely overlap withexisting targets rather than being deposited on areas with approximatelybackground levels of signal. For a camera with 5 megapixels, and atarget that spans approximately 2 pixels, this corresponds to a dynamicrange that spans roughly from 10 to 250,000 targets, or a range span of25,000. This range is adequate for many applications, and in thoseapplications for which a greater dynamic range is required, multipledilutions can be used.

In a second mode, the differences between a single target and differentnumbers of targets within a pixel can be discriminated. For example, ifthe signal is measured with an 8-bit pixel, with 256 levels, and abackground signal is 12, then a single binding event might average 62,two targets in the same pixel might average 112, and so on. In thiscase, the dynamic range is far higher, and is roughly the number ofpixels times the number of levels that can be discriminated divided bythe average number of pixels per target (with a floor of one) andfurther divided by a factor of approximately 10, representing thesaturation at which additional target binding could raise levels in asignificant number of pixels above the pixel saturation level. In thiscase, with 5 levels being able to be discriminated and an average numberof pixels per target being 1, the dynamic range is still roughly aminimum of 10 (limited by solely statistical considerations), but theupper level now extends to approximately 2.5 million, or an additionalten fold dynamic range from the previous example. The difficultyencountered with this second mode of operation is that it becomesincreasingly difficult to distinguish specific from nonspecific bindingon the basis of image analysis—both because on average each target spansa smaller number of pixels, and because the contrast between differentlevels is generally poor.

While these methods can distinguish individual binding events, it shouldbe noted that the greatest value of counting individual binding eventsoccurs when there is significant nonspecific binding or other forms ofnoise. For example, low level background noise can sum over a large areato comprise a large noise signal, for which a large amount of specificsignal is required to show above background. However, in cases where thesignals are generally large above background, it can be convenient touse a signal summing method, wherein the signal is summed either byadding the signal values at each pixel, or by using an analog summingtechnique such as the use of a photodiode or a photoresistor or aphotomultiplier tube (PMT).

Controlled Washing Dynamics

In the following discussion, electric potential between the electrodes,and through potential the resultant electrophoretic force, is used as anexample of a vertical force on the target 114. It should be noted,however, that the modulation of force can also be effected in similarmanners when using other means of force application, such as magneticforce (e.g. on tags 270 comprising magnetic particles). In addition, theforces can also include horizontal forces, applied such as through theapplication of lateral electrophoresis, lateral application of magneticfields, and different means of application of lateral forces. It shouldbe noted that the application of these lateral forces can also includethe introduction of an air bubble or similar air-water interface, sincethese interfaces apply very large forces through surface tension ontargets 114. It should be noted that the application of these forces canbe shaped, so that the forces are neither vertical nor horizontal, butcan have aspects of both, such as shown in the FIG. 5.

As described in FIGS. 28A and 28B, the washing force is increasedincrementally over time, with realtime detection of binding occurring asdescribed above. The dynamics of changing the washing stringency is donein fixed incremental steps in its simplest form. FIG. 28A is a graph ofthe washing potential as a function of time for a simple step washingfunction. The horizontal axis is time, and the vertical axis is thepotential of the probe electrode 200 relative to that of a referenceelectrode. At the initial period, the potential is zero or low, in whichcase there is no washing force. The detected signal represents the sumtotal of the specific and the nonspecific binding of tag target to theprobe 116. After a time T9, the negative potential on the probeelectrode 200 is increased by a value V1 in rough step function. Aftersome period at this higher potential, generally on the order of hundredsof milliseconds (though as small a period as tens of milliseconds and aslarge a period as seconds), realtime detection occurs. The step increasein the negative potential is repeated a number of times until themaximum required potential occurs. The maximum potential will generallybe currently determined as the force with which all specifically boundtag target is released from the probe 116. The number of steps ofpotential increase is user selected, and will be determined by factorssuch as the range of different binding forces between tag targets andprobes 116 present on the electrode 200. It should be noted that thepotential steps need not be equal in size, nor do the time periods T9need to be necessarily equal as well.

The binding between the tagged target 275 and the probe 116 can becomplex.

Consider FIG. 29A, a schematic diagram of a tagged target 275 comprisinga single-stranded DNA target 470 binding to a complementary DNA probe480, which is bound to the substrate 120 at a single point of attachment117. As can be seen, electrophoretic force exerted on theelectrophoretic tag 270 will tend to unravel the DNA target 470 from thecomplementary DNA probe 480 in a straightforward manner. FIG. 29B is aschematic diagram of a tagged target 275 comprising a single-strandedDNA target 470 binding to a complementary DNA probe 480, which is boundto the substrate 120 at multiple points of attachment 117. In this case,force exerted on the electrophoretic tagged 270 does not necessarilydirectly result in release of the target 470, because the target 470 isconstrained by the probe 480 within the points of attachment 117.Because of the topological constraints and the multiple points of force,the target 470 must be gently removed from the probe 480. This can beaccomplished by various means as described below.

It should also be appreciated that the electrodes of the cell canpotentially participate in electrochemical reactions that limit thepotentials at which the cell can be operated. For example, if theelectrodes comprise indium tin oxide, potentials of just over 1 volt canresult in deterioration of the electrode. If larger voltage potentialsare required in order to separate the target 114 from the probe 116, thevoltage potentials can be applied for a very short period—which can beten of milliseconds, and even more preferably one millisecond, and evenmore preferably 100 microseconds, during which time the target 114 canbe separated from the probe 116 but which is of such a short durationthat relatively little reaction of the electrode material can occur. Inintermediate periods, wherein a voltage potential is maintained thatdoes not cause electrochemical reactions involving the electrode, theamount of target 114 attached to the probe 116 can be measured.

FIG. 28B is a graph of the washing potential as a function of time for aramped washing function. In this case, the overall potential betweendifferent steps of washing is the same potential difference V1 as usedin FIG. 28A. However, instead of a step function, the potential isgradually raised over time. The increase in potential can be linear,exponential, or otherwise. Alternatively, the increase in potential neednot be monotonic, and can comprise a series of increasing and decreasingsteps arriving at the desired potential for the intended stringency.

Furthermore, the intermediate potential “plateaus” are divided into twodiscrete time steps. In a first time step, TD, the target 114 and theprobe 116 are allowed to dissociate at the higher potential. However,the rate of dissociation during the detection process is desired to bereduced, so that the potential can be reduced during a time TC (e.g.during image capture) while the bound target 114 is detected.

The direction vector of the electric field used for washing need not bein one direction, but can be usefully varied to help remove the target470 from the probe 480, as illustrated in FIGS. 30A and B. FIG. 30A is aschematic side view diagram of two reference electrodes E10 and E11relative to the probe electrode E12. FIG. 30B is a graph of thepotential of electrode E12 relative to the two reference electrodes E10and E11 as shown in FIG. 30A for two steps in the washing stringency.The relative potential of electrode E10 to electrode E12 is given by adotted line, the relative potential of electrode E11 to electrode E12 isgiven by a dashed line, and wherein the relative potentials overlap, adashed-dotted line is shown. For an initial period, the potentialrelative to electrode E10 is high, and the potential relative toelectrode E11 is zero. Next, the potential relative to electrode E11 ishigh, while the potential relative to electrode E10 is small. Next, thepotentials for both electrodes E10 and E11 are placed at an intermediatelevel. During these three steps, the electric field direction variesfrom pointing at the electrode E10, to the electrode E11, to a positionintermediate between the two. Target 470 that is sterically enmeshedeither with the probe 480, or alternatively with the linkers 118, orother material that is at the surface 120, can generally be pulled inthe direction that will release it, whether it is bound specifically ornonspecifically. In subsequent time steps T10, as shown in the figure,higher forces in the different directions can be applied. In addition,instead of applying the forces as step functions, they can be applied asramped time functions (linear, exponential, or other) and they can alsobe non-monotonically applied and applied over varying durations toprovide the desired effect.

It should be noted that the potential needed to separate the taggedtarget 275 from the probe 116 depends on the charge on theelectrophoretic tag 270. Furthermore, it can be less convenient if thepotential needed to be applied varies over large orders of magnitudeover the probes 116 affixed within an array 180, since a larger numberof different stringency washes will be required. Thus, it is convenientif the electrophoretic tag 270 is matched roughly to the binding forcebetween the associated target 114 and probe 116. For instance, atarget-probe pair with a stronger binding force will be convenientlypaired with an electrophoretic tag of larger electrostatic charge, sothat more roughly the same voltage potential would need to be applied toseparate the target-probe pair.

While the washing dynamics of the previous section deal withelectrostatic or electrophoretic forces, other discriminating forces andconditions can be applied with the use of realtime detection, includingconductance, pH, solvents, and competing ligands. Such conditions canfurther be applied either in a stepwise fashion, or in a continuousgradient, and can be applied using mechanical pumps, electroosmoticmechanisms, or other transport mechanisms. Furthermore, these conditionscan be applied in combination with each other, or also in combinationwith the electrostatic and electrophoretic forces described above.Because it is difficult at times to reproducibly change, for example,the pH of a solution in a gradient fashion, especially in the very smallformats used in many of the assays of the present invention, it isuseful to place within the array 180 a number of target 114-probe 116pairs whose binding is disrupted at known conditions, serving thereby asinternal controls to verify that specific conditions are being reached.

Detection of Organisms and Determination of Anti-Organism AgentSensitivity

Overview

It is important to determine the identity of bacteria with regards tofood pathogens, biological warfare agents, and a variety of animal andhuman diseases. In addition to the rapid and sensitive detection ofthese bacteria, in the case of animal and human disease, it is of greatbenefit to additionally determine the susceptibility of themicroorganisms to antibiotics, antifungals, and other medical agents.Bacteria that are of particular interest are human pathogens, includingbacteria from the genera Pseudomonas, Stenotrophomonas, Acinetobacter,Enterobacter, Escherichia, Klebsiella, Proteus, Serratia, Haemophilus,Streptococcus, Staphylococcus, Enterococcus, Mycobacterium, Neisseria,and other human pathogens encountered in medical practice. The presentinvention is well suited to this application.

It should be noted, however, that the detection system and methods arenot limited to bacteria, and can be used as well in the detection offungi (e.g. Candida and Aspergillus), virus, mycoplasma, and other typesof organisms, and can include the detection of animal cells, such as inthe detection of metaplastic or other disease cell types. In thediscussion below, the use of bacteria is meant to be as an example only,and that the discussion is to include these other organisms as well. Itshould be noted that in the discussion above, the target 114 can begenerally and without limitation bacteria and other organisms, asdiscussed below. The discussion below expands the detail and introducesnew methods and devices with which the application of the methods anddevices above are applied to bacteria and other organisms.

FIG. 31 is a block flow diagram of the process for determining theidentity, number and antibiotic susceptibility of bacteria in a sample.In a first step 700, the sample is optionally concentrated, which isnecessary in many cases where the bacterial sample is present in a largeliquid volume. Such a step 700 will generally be performed when thesample is in a volume of greater than 10 milliliters, and often when thesample is in a volume of as little as 500 microliters. The reason forthis is that, depending on the system, the sample volume to be placedinto the detection system can be limited to as little as 100microliters, although other systems can handle much larger amounts, withsamples in the many milliliters. The concentration performs twofunctions. First, the ratio of number of bacteria to the volume of thesample is increased, so that the greatest possible fraction of thesample can be used in the system. A second reason is that the bacteriamay be in a liquid whose electrical or other properties are incompatibleor non-optimal for the detection system. For example, if electrophoreticmethods are subsequently to be used, the efficacy of such methods isimproved generally by the use of low-electrolyte buffers. In such case,the bacterial sample liquid will be replaced by a liquid that is morecompatible with the system.

In a step 710, the bacteria are transported to a detection zone, whichis where the locator for use in later steps is located. The bacteria atthis point are still not immobilized, but are free to move about in athree-dimensional volume. The transportation to the detection zone canbe accomplished in many different ways, including active physicaltransport by pumps (e.g. positive displacement syringe pumps, pneumaticpumps, peristaltic pumps, or others), by electroosmosis, by gravity, byelectrophoresis of the bacteria, or other means. In addition, theconcentration step 700 can involve the concentration of the bacteriadirectly in the detection zone, such as centrifugation of the bacteriainto the zone, followed by resuspension.

In a step 720, the bacteria are immobilized onto the detection surface.The detection surface, it should be noted, can either be specific for asubset of bacteria (e.g. through the surface attachment of antibodiesspecific for one or more bacteria), or it can alternatively benonspecific such that most or all bacteria in a sample will bind to thesurface.

It should be noted that there can be a single zone or multiple zoneswithin the system. For example, in one embodiment of the system, therecan be a zone specific for each bacteria strain—distinguished by theproperties of the respective detection surfaces—and the system cancomprise even dozens of separate zones. Alternatively, all of thebacteria can be attached at a single nonspecific zone. Also, there canbe a combination of specific and nonspecific detection zones, where thebacteria are first captured at specific detection zones, to be followedby nonspecific capture of all of the bacteria that were not captured inthe specific detection zones. It should be noted that if there aremultiple zones, the bacteria may need to be transported from onedetection zone to another detection zone between attachments of thebacteria to the respective detection surfaces.

Now that the bacteria are attached to the surface, their arrangement isroughly in a two-dimensional distribution. It should be noted that theterm two-dimensional is meant to include some reasonable degree ofvertical depth, given that the attachment surface can be up to micronsin depth (e.g. through the incorporation of polymer hydrogel or similarmaterials). However, given that the detection means can incorporatemicroscopic detectors with limited depth of field, it is preferable forthe surface to have a topological depth of no more than 5 microns, andeven more preferable for the depth to be less than two microns.

The attachment of the bacteria to the surface can occur throughdiffusion of the bacteria to the surface, where they are attachedspecifically or nonspecifically. However, because diffusion can be slowon the time scales generally desirable in such a system, active meansare preferred for the attachment of the bacteria. These means cancomprise the use of electrophoresis or dielectrophoresis of the bacteriato the surface, the use of centrifugal forces, or even the filtration ofthe bacteria onto the detection surface, wherein the surface is porousbut with pores smaller than that of the smallest diameter of thebacteria. Once the bacteria are in direct contact with the surface, itis generally arranged that the attachment process is rapid, occurring ina matter of seconds or minutes, whether the attachment is specific ornonspecific, although longer times of attachment are allowed within thepresent invention. It should be noted that bacterial attachmentgenerally increases over a period of time (minutes to hours), both withthe secretion of attachment molecules from the bacteria, as well as anincrease in the number and strength of attachments that normally accrueeven with the attachment of non-living material to surfaces—however, theattachment above is meant to indicate attachment such that the typicalforces of diffusion, convection, fluid flow through the system, and suchare insufficient to dislodge the bacteria from the surface, and thatspecific application of forces desired to remove the bacteria isrequired.

In cases where specific attachment of bacteria is desired onto thedetection surface, it is useful to remove those bacteria that arenonspecific attached to the surface. This is generally accomplished bythe assumption that all of the bacteria that are attached specificallyare attached with a relatively narrow range of attachment forces. Thus,forces outside of that specific-attachment range will either remove thenonspecifically attached bacteria (i.e. those bacteria that are attachedwith a lower force) or will remove specifically attached bacteria, butnot others (i.e. those bacteria that are attached with a greater force).The types of forces that can be employed to this effect includeelectrophoresis, dielectrophoresis, centrifugal force, hydrodynamicforces (i.e. fluid flow across the surface) and other means of applyingspecific forces. In addition, the strength of the specific ornonspecific binding can be altered by changing the characteristics ofthe medium, such as conductance and pH.

In a step 730, the number of bacteria in each detection zone, attachedto the respective detection surface, is detected via automatic means. Ingeneral, the means of counting bacteria will be through automatic visualinspection of images taken of the detection surface using magnifiedimages, or through measurement of spectral intensity or scattered lightintensity. Because of the lack of refractive index contrast betweenbacteria and the surrounding medium, the detection of the bacteria canbe enhanced via techniques well-known in the prior art, including theuse of phase contrast, differential interference contrast, fluorescenceor other means.

It should be noted that the identification of the serotype, strain,species, genus, or other specific typing of the bacteria has beenaccomplished to the extent that the attachment of the bacteria to thedetection surface is specific. However, if the attachment surface isnonspecific or broadly specific (e.g. having specificity for a range ofbacterial types), the identification can be at this moment incomplete.The use of stains, which can include the use of specific antibodies withoptical tags (e.g. fluorescence, scattering, absorption) or tags thatpermit other forms of detection (e.g. chemiluminescence, radioactive,redox, conductivity or other modes of detection), can be optionally usedat this point to determine the type of bacteria attached to thedetection surface.

It should also be noted that at this point, not only are the numbers ofbacteria determined by the system, but that the specific locations ofthe bacteria with respect to the detection surface are also known.Because the type of the bacteria are also known (because of attachmentto a specific surface or because of the use of a specific stain), eachbacterium is now associated with a location and a type. With the tightattachment of bacteria, this information will be relatively constantthrough the operation of the system. The location noted above caninclude both the location of an individual bacterium, as well as thelocation of clusters of bacteria, that can represent roughly sphericalclumps as well as linear chains of bacteria.

It should further be noted that in order for the number of bacteriadetected by the system to be accurate, it is preferable for at least 50%of the bacteria in the original sample of the step 700 to be attachedcumulative to one or more of the detection surfaces, and even morepreferable for more than 80% of the bacteria to be attached. The use ofthe active transport of the bacteria to the detection surface in thestep 720 is an important aspect of this accuracy.

For use in disease diagnosis and treatment, it is of great benefit toknow not only how many bacteria are present, but also to determine theviability of the bacteria, and also their susceptibility to differentantibiotics, singly and in combination. The following steps areoptionally employed depending on the desired information generated bythe system.

In a step 740, the viability of the bacteria on the detection surface isdetermined. In general, this is performed in one of two means. In afirst means, the detection surface and the bacteria thereon areincubated in the presence of a growth medium, which allows the bacteriato grow and divide. Any bacterium that can be visually determined toengage in growth and division is then indicated as viable. In a secondmeans, vital and mortal stains can be employed to detect bacteria thatare viable or non-viable. It should be noted that the total number ofbacteria is equal to the sum of the viable and non-viable bacteria, sothat the use of any two measures of total bacteria, viable bacteria, andnon-viable bacteria will allow the calculation of the third measure.

It should be noted that certain organisms that would be detected in themanner of the present invention may not be viable by themselves, but mayrequire a host (e.g. for the detection of a virus). In that case, thedetection surface can comprise host cells that support the growth of thevirus or other organism. In that case, the step 730 of counting thebacteria would be replaced by a step in which the number of infectedhost cells would be counted. Such step of counting is accomplishedaccording to the characteristics of the virus and the host, and caninclude the presence of cell surface markers indicative of infection, bychanges in the physiology of the host that results from infection, orthrough lysis or death of the host.

In a step 750, antibiotic in a medium supporting growth can beintroduced into the medium of the detection zone, so that the bacteriaare then in the presence of the antibiotic during growth. It should benoted that if the organism being detected is not a bacterium, thetreatment is matched to that of the organism. Thus, the detection offungi or yeast would be matched with the use of antifungal agents, andthe detection of viruses would be matched with the use of anti-viralagents. The bacteria are kept in the presence of the antibiotic fordifferent times and concentrations of clinical interest, and the steps730 and 740 are repeated after an appropriate incubation period orperiod of effect (i.e. the time for the agent to take effect, whichcould take place in the absence of the antibiotic). Repetition of thesteps 730, 740 and 750 can be performed in order to test theeffectiveness of different agents, or different treatment regimens.

The methods and system of the present device will now be described inmore detail.

Sample Concentration

Samples can range from a milliliter up to a liter for certainrespiratory lavages, and can further range in bacterial concentrationfrom 10 bacteria to greater than 10⁶ bacteria per milliliter.Furthermore, the sample can be present in blood, urine, sputum, lavagefluid or other medium. Sample concentration both concentrates the sampleso that bacteria that are present in small numbers can all beeffectively introduced into the system, as well as so the backgroundliquid medium can be normalized to have consistent properties uponintroduction to the system. It should be noted, however, that certainsamples, can be used without concentration or other modification withinthe present invention.

Conventional methods of sample preparation in the prior art can be usedfor this purpose, including filtration and centrifugation, followed byresuspension of the bacteria in a small fluid. It should also be notedthat centrifugation can be accompanied by flocculation, precipitation oraddition of a co-precipitate, and such methods are encouraged in thatthey permit the handling of very small numbers of bacteria, and preventaggregation of the bacteria. In any of these cases, however, it ispreferable that no material be added that will remain a particulate,especially with properties (size or density) similar to that of thebacteria (e.g. the use of polymer beads).

Another method in accord with the present invention is the use ofcollection with an elutable collector. In such a system, the sample isfiltered through a matrix which is densely packed with a material thatnonspecifically binds bacteria. This material has the further propertythat the property that binds the bacteria can be reversed throughchemical, enzymatic or physical means such that the bacteria can beeluted from the material subsequent to bind. Such a collector can beused both to place the bacteria into a uniform medium that is wellsuited for further steps in the method, as well as to removecontaminating material that has size or charge differences from thebacteria that are desired to be monitored.

A preferred embodiment of this sample preparation is that of a cartridgewith volume of 50-1000 microliters, and preferably less than 250microliters, in which an ionic exchange resin, is packed. This resin isconveniently supplied in bead form, and can either be permanentlycharged (e.g. through the use of quaternary amines) or reversiblycharged (e.g. through the use of a secondary or tertiary amine).Furthermore, the size of the beads (or pore size of the resin) should besuch that in the absence of the charge group, the bacteria would floweasily through the interstices of the bead, and that flow rates throughthe beads will be reasonable according to the volume of the sample (thebeads will preferably be greater than 10 microns in diameter, and lessthan 2000 microns, and more preferably greater than 20 microns and lessthan 1000 microns and even more preferably greater than 50 microns andless than 500 microns).

In this preferred embodiment, the sample can be pressed through thecartridge either without modification, or with the addition of a bufferto regulate the pH, and/or also in the presence of a preferablynon-ionic detergent, in order to reduce nonspecific binding of thebacteria to the system components or to each other. It is preferable forthe pH to be relatively neutral (in the range of pH 6 to 8), and in anycase sufficient that the bacteria maintain a negative charge, and thatthe resin maintain a positive charge. This negative charge is typicalfor most bacteria, but it should be noted that for any organism that istypically positively charged, a cationic resin can be substituted forthe anionic resin, and the control of pH will be the opposite of what isdescribed above and below for negatively charged organisms. Due to theopposite polarities of the organism and the resin, bacteria that passclose to the resin will be captured by electrostatic interactions to itssurface and stick. This serves to concentrate the large bacteria from alarge volume to that of the volume of the cartridge.

In order to release the bacteria from the resin, the pH of the solutioncan be changed so that the interaction of the resin and the bacteria isreduced. For example, at a high pH (i.e. above the pK of the cations onthe anionic resin), the cations on the resin lose their charge, andtherefore their relative ability to capture the bacteria. Alternatively,at a low pH, the anions on the bacteria giving rise to their negativecharge are protonated, and therefore lose their attraction to the resin.

It is important to find conditions under which the bacteria bind, andothers in which the bacteria can be released. In order to mediate thestrength of attraction of the bacteria and the resin, other factors thatcan be modulated include the ionic strength of the solution (i.e.counter ions will tend to reduce the electrostatic attraction), thecation functional group that is used on the anionic exchange resin (e.g.primary, secondary, tertiary or quaternary amine), or the density of thecations on the surface of the resin (i.e. reducing the density willgenerally reduce the attraction of the bacteria to the resin).

The bacteria can in general be eluted from the resin in a volume notsignificantly different than that of the cartridge, and with care takennot to mix the eluting solution, even smaller than that of thecartridge. In general, after elution from the cartridge, the solutionwill be neutralized, preferably with a zwitterionic buffer so that theconductance of the buffer is not increased too much. Other properties ofthe resulting medium can be adjusted as needed, including ionicstrength, conductance, the presence of surfactants, the presence ofnutrients or growth factors for the bacteria, and the pH. In general, aswill be discussed below, it is preferable for the bacteria to be inrelatively low conductance solution. Given that the elution will beperformed at pH's either above 3 or below 11, the resulting neutralizedsolution is likely to have an ionic strength of less than 10 mM salt,which is preferable for the subsequent steps.

It is also convenient as part of or prior to the concentration step toperform a pre-filtering in order to remove larger contaminants, whileallowing the passage of the bacteria to be monitored. Such filters cancomprise nitrocellulose, nylon, cellulose, or other membranes, beadfilters, or other filters as may be convenient. It is also within thespirit of the present invention for the elutable collector above toserve both as an ion exchange resin as well as a size filter. Even incases where elutable collectors are not used, it is still convenient touse a size filter to remove non-bacterial contaminants. Furthermore, itis also convenient, depending on the source of the sample and the natureof the contaminants, to use a size filter that removes contaminantssmaller than the bacteria in the sample; this may not be a problem forthe detector, but the smaller contaminants can compete with the bacteriafor spots on the surfaces to which the bacteria are meant to attach,reducing the attachment of the bacteria.

Transport to the Detection Zone and Attachment to Detection Surface

The detection zone is conveniently within an enclosed cell, andcomprises one or more surfaces on which bacteria will be immobilized anddetected. A general format for a detection cell is shown in FIG. 32A,which is a top schematic diagram of a bacterial detection cell 804, andin FIG. 32B, which is a side view schematic diagram of the bacterialdetection cell of FIG. 32A through the cross-section X.

The cell 804 comprises two chambers 805, of which there can be as few asone and tens or even hundreds. Each chamber will be used either tohandle a different bacterial sample, or to handle side-by-side a singlesample, in which the bacteria will be treated with different growthmedia, antibiotics or other anti-organism agents, antibioticconcentration profiles, temperatures, or other physical, chemical orbiological conditions to which the bacteria will be subjected. Thechambers 805 are shown as enclosed on all sides, but it is consistentwith the present invention for the chamber to be open, such as in aformat of a microtiter plate well. If the chamber 805 is closed, aninput port 803 and an output port 802 are provided for changing thesolution within the chamber 805.

The size of the chamber 805 can vary within the spirit of the presentinvention, but it is preferable for the width to be 200-5000 microns,and more preferably 500-2000 microns, and most preferably 500-1000microns, and it is preferable for the height (i.e. the distance betweenthe electrodes) to be 100-2000 microns, and more preferably 200-1000microns, and most preferably 250-500 microns), and it is preferable forthe length to be preferably of 0.5-20 mm (depending, in part, on thenumber of capture zones, as will be discussed later). These dimensionsare primarily related to the fluid handling (e.g. the larger the volume,the easier it is to handle larger sample volumes), the detector optics(e.g. if it is desired to see individual bacteria, then themagnification must be of a certain amount, which lowers the field ofview), the rate at which bacteria can be moved vertically (e.g.depending on the flow of the bacteria through the chamber, the rate ofmovement must be large enough to allow deposition on the proper surfacesbefore the bacteria leave the cell), and the dynamic range of thedetector (e.g. the number of bacteria can “lie flat” on the surface andbe distinct in the detector).

The application of microfluidics devices is well-known in the art, andcan be seen, for example, in the services and products of Micronics,Inc. of Redmond, Wash., and CFD Research Corporation of Huntsville, Ala.These devices can handle very small amounts of material, which can befractions of a nanoliter, and which comprise components which can havemultiple functions including sample injection, microdispensing,concentrators, multiplexers, separators, sensors, pressure-driven flow,electroosmosis, electrophoresis, dielectrophoresis, particle transport,electrochemical sensing, electromagnetics for moving paramagneticparticles, and more. These microfluidics technologies are well suitedfor the present invention.

In the chambers 805, an anode 816 and a cathode 815 are used to create azone in which placing a potential on the electrodes 816 and 815 will, inthe presence of a suitable buffer, cause electrophoresis to occur. Whilenot required, it is preferable for the electrodes 815 and 816 to beparallel, on opposite walls of the chamber, both in terms of ease ofmanufacturing, as well as causing the electrophoretic fields that willbe generated to be perpendicular to the surface of the electrodes andresulting in even movement of bacteria to the respective electrodes. Atleast one of the electrodes 815 or 816 will be largely transparent, tothe extent that bacteria can be detected through the electrode by visualdetection means, as will be described below. Transparent conductivesurfaces that can serve as the transparent electrode include ITOsputtered films, and printed transparent conductive inks, such as theS-100 and P-100 inks provided by Sumitomo Osaka Cement. Thenon-transparent electrode can comprise evaporated metallic coatings(gold, silver, aluminum), but the preferred electrode material isplatinum or other refractory metals, which can be plated by variousforms of chemical or physical vapor deposition.

On the anode 816 are placed capture surfaces 820, on which bacteria willadhere. These capture surfaces will have capture agents with specificaffinity for different bacteria, although some of the capture agentswill, as will be described later, have general affinity for bacteria orfor large groupings of bacteria. For specific affinity, the affinity isgenerally provided by antibody preparations, which can be polyclonal ormonoclonal, with specificity for a small number of bacteria, or canalternatively be provided via aptamers, or other specific affinitymolecules. A loading surface 810 is also present, on which bacteria canoptionally be concentrated prior to movement to the capture surfaces820. The loading surface 810 can have a weak or reversible attractionfor bacteria, which will dwell on the surface 810 for a period of time,or the loading surface 810 can have very low specific or nonspecificattraction for bacteria. Alternatively, the loading surface 810 can haveno attraction for bacteria, but will be held close to the loadingsurface via electrophoretic fields, as will be discussed below. Ingeneral, other surfaces of the chamber 805, including those areas of theanode 816 between the loading surface 816 and the capture surfaces 820,or between the different capture surfaces 820, will have very lowbinding to bacteria, such as that provided by OptiChem coatings (Accelr8Technology Corporation, Denver, Colo.).

This low binding is generally conferred by a coating applied to theelectrodes 815 and/or 816, wherein the coating preferably has componentsof polyethylene glycol, polyacrylamide or other low surface energypolymer. Preferably, this polymer has been functionalized (e.g. withN-hydroxy-succinimide, thiol, epoxy, hydrazine, or amino groups, or withbiotin or avidin) such that agents that bond specifically ornonspecifically to bacteria can be attached, so as to confer upon thecapture surfaces their attractive characteristics with bacteria.

Bacteria can exhibit high nonspecific binding after contact with arelatively non-attractive surface, especially after being in contactwith that surface for a period of time. Some of this binding comes withthe expression of bacteria attachment proteins, and can include, forexample, various adhesin proteins. In order to reduce the amount ofnonspecific binding, in those cases where nonspecific binding is notdesired (e.g. to the loading surface 810, or as bacteria are being movedbetween specific capture surfaces 820 to which they do not normallybind), it can be convenient to use various agents that can reduce thisundesirable nonspecific binding, including the use of blockingantibodies that bind to the adhesins, the use of various adhesin-bindingagents, such as galabiosides, globotetraoses, and tetrasaccharides, orthe use of various detergents (and preferably non-ionic detergents) toreduce this binding. In addition, the binding of the bacteria tosurfaces is responsive to both the time of residence, as well as theforce with which the bacteria are directed onto the surface. By reducingthe electrophoretic force, or by reducing the time over which thebacteria are directed to the electrode by electrophoresis, the force ofnonspecific binding can be modulated. In addition, it has been foundthat placing a charge on the electrode that has the same polarity ofthat of the bacteria can also reduce the nonspecific binding.

The attraction of the capture surface 820 for bacteria can be highlyspecific or relatively nonspecific, regarding the type of bacteria. Forexample, the surface 820 can comprise nonspecific polycationic polymers(e.g. polyethyleneimine or polylysine), antibodies specific forserotype, genus, species or class, aptamers, glycoprotein-bindingproteins, or others.

While it is shown that there is only the single cathode 815 and thesingle anode 816, it is also within the spirit of the present inventionthat there can be multiple electrodes, which can be separatelyaddressable, especially in the case of the anode 816. In such case,individual anodes 816 can be placed roughly at the same locations as thedifferent capture surfaces 820. In the following discussion, where it isindicated that bacteria are being electrophoretically transported to aparticular capture surface 820 or loading surface 810, or a force isbeing directed away from said surface, this can be accomplished eitherby activating the single electrode 815 and 816 as shown, oralternatively by activating separate electrodes that lie underneath therespective surfaces. FIG. 32C is a side view schematic diagram of thebacterial detection cell of FIG. 32B with the use of addressableelectrodes. It should also be noted that the use of these addressableelectrodes can be used to create horizontal electrophoretic forces, suchthat bacteria that are bound to the loading surface 810, for example,under the influence of an addressable electrode 817A and an addressableelectrode 819A, can be moved to the first capture surface 820 by placingboth electrodes 817A and 819A under a relative negative potential, aswell as the electrode 817B, while placing the electrode 819B at arelative positive potential, such that the electrophoretic force fieldlines transport the bacteria from the loading surface 810 to the firstcapture surface 820. It should be noted that there are a number ofdifferent arrangements of electrodes that would have similar effects,including the use of addressable cathodes 817 and addressable anodes 819that are offset from one another horizontally, or that there are amultiplicity of addressable cathodes 817 that are activated to differingdegrees in order to shape the electrophoretic force fields so as toprovide a relatively even distribution of bacteria on the capturesurface 820. It can also be beneficial in certain circumstances to havethe bacteria distributed in a non-uniform manner on the capture surface820. For example, in the case where the number of bacteria can rangeover numbers larger than the nominal range of the system with uniformdistribution of bacteria, by having a non-uniform distribution on thecapture surface 820, areas of relative paucity of bacteria can be usedwhen the number of bacteria in the sample is high, whereas areas ofrelative concentration can be used when the number of bacteria in thesample is low.

It should be noted that the vertical distances represented by theelectrodes 815 and 816 and by the surfaces 810 and 820 are not drawn toscale. While the separation between the electrodes will generally behundreds of microns, the vertical dimensions of the electrodes 815 and816 will generally be measured in tens of nanometers, and the surfaces810 and 820 will be nanometers to tens of nanometers thick. The size ofthe surface 810 and 820 can vary greatly within the present invention,but are preferably hundreds of microns up to 5 millimeters in eitherdimension of the top-view diagram. Likewise, the distance separating thesurfaces 810 and 820 can vary greatly, but will preferably be between 5microns or as large as 1 millimeter, and more preferably be between 50and 200 microns.

FIGS. 33A-D are side schematic views of the transport and capture ofbacteria using the chamber of FIGS. 32A-B. In FIG. 33A, bacteria of twotypes (denoted by stars 830 and 835 and diamonds 840) are introducedinto the chamber 805 via the input port 803 (the difference betweenbacterial symbols that are filled or open will be described below).

A potential is placed across the anode 816 and the cathode 815 such thatelectrophoresis between the two electrodes is created. Thiselectrophoresis can be accelerated by the use of chemical agents asdescribed above. Optionally, an additional cathode can be placed outsideof the port 803 to create an injection field that promotes the movementof bacteria into the chamber 805, as will be discussed in more detailbelow.

As bacteria 830, 835 and 840 move past the beginning of the anode 816,they move towards the anode on the basis of their generally negativecharge. It should be noted that the negative charge of the bacteria can,to some extent, be modified by the pH of the medium in which theelectrophoresis takes place. To the extent that some bacteria may have aneutral or slightly positive charge in a medium, it is convenient toraise the pH of the medium so as to confer on the bacteria a morenegative charge.

The movement of the bacteria 830, 835, and 840 is at a speed dependenton many factors, including the potential between the electrodes 815 and816, the conductance of the medium, and the presence of chemical agentsto accelerate the electrophoresis, and simultaneously, there is movementof the medium through new medium (with or without bacteria) into theinput port 803 and out of the output port 802. As mentioned above, theuse of addressable electrodes 815 and/or 816 can be used to promotemovement of the bacteria, as well. The balance of vertical movement(e.g. via electrophoresis) and horizontal movement (e.g. via fluidmovement, electrophoresis, or other means) should be such that thebacteria will contact the loading surface 810. On the loading surface,the bacteria can either be prevented from horizontal movement either byweak electrostatic forces (e.g. via a weak electrostatic charge on thesurface) on the loading surface 810, or will show reduced movement dueto lower fluid flow near to the surface in the presence of anelectrophoretic force downwards to the loading surface 810. These forceswill generally be orthogonal or nearly so, which is convenient sincethis allows independent adjustment of the movement of the bacteria inboth vertical and horizontal directions. In those cases where greaterhorizontal movement is necessary, for example, a larger vertical forcecan partially or entirely compensate.

The purpose of the loading surface 810 is to place the bacteria into avery small volume on the capture surface, as shown in FIG. 33B, and tocompensate for a dilute sample. Once all of the bacteria are collectedonto the surface 810, then their movement onto specific capture surfaces820 is more easily accomplished.

In FIG. 33C, the bacteria are moved from the loading surface onto aspecific capture surface 820. If the loading surface 810 has anelectrostatic attraction for the bacteria, the electrostatic force isreversed, as will be discussed in more detail below. If the bacteria areheld close to the loading surface 810 solely by virtue of theelectrophoretic field, this field is either turned off, reduced, or evenreversed.

The bacteria are then moved horizontally along the chamber 805 throughmovement of the fluid, which movement may be accomplished viaelectroosmosis, positive displacement pumps, peristaltic pumps, or othermeans. This movement is coordinated with the further application ofvertical electrophoresis, which coordination can be simultaneous orsequential. That is, in sequential coordination, fluid movement can beperformed for a certain period, and then followed by a period of fluidnon-movement during which electrophoresis is applied, or theelectrophoresis can be applied during movement in simultaneouscoordination. In the latter case, the speed of movement or the magnitudeof the electrophoretic force can be varied, such that bacteria do notmove more than the width of a capture surface 820 before contacting thesurface 820. Indeed, it is preferable that all of the bacteria do notcontact the capture surface 820 at its leading edge (i.e. to the left inthe figure), so that there is a more even distribution of bacteria onthe capture surface 820.

Instead of the bacteria 840 moving horizontally across the chamber 805,the bacteria can alternatively be moved in a “zig-zag” fashion ifaddressable electrodes corresponding to the various surfaces 810 and820A-E are used. That is, the bacteria 840 can be moved from the loadingsurface 810 to the electrode 815 by the proper potential being placed onthe addressable electrode beneath the loading surface 810, andafterwards, the bacteria 840 can be moved to the first capture surface820A by placing a positive potential on the electrode beneath thesurface 820A and a negative potential on the electrode 815. Then, thebacteria 840 can be successively moved from the capture surfaces 820A-Eto the electrode 815 and thence to the next capture surface 820B-E. Thishas the advantage that bacteria 840 do not accumulate on the trailingedges of the various capture surfaces 820 (i.e. stick to the first partof the surface that they encounter), but rather are more evenlydistributed on the capture surfaces 820. This effect can be furtherstrengthened by using addressable electrodes replacing the singleelectrode 815, wherein these addressable electrodes can either bedirectly on top of the corresponding addressable electrodes beneath thecapture surfaces 820, or alternatively can be staggered with respect tothe capture surfaces 820 in the horizontal direction.

As can be seen in FIG. 33C, on the leftmost capture surface 820A, onlythe bacteria 840 are bound, whereas the bacteria 830 and 835 do not bindat this surface. In FIG. 33D, as the process is repeated and thebacterial sample is brought into contact with additional capturesurfaces 820, the bacteria 830 and 835 are now attached to the capturesurface 820C.

An alternative embodiment is shown in FIGS. 34A-D, which are side-viewschematic diagrams of electrophoretic transport to the detectionsurfaces. In FIG. 34A, a bacterial sample in low-electrolyte medium 882is brought into contact with a high-electrolyte medium 880 with aninterface 890 formed between them at roughly the location where a sampleinput port 895 intersects the chamber, which has an alternative inputport 896. This interface can be formed by movement of thelow-electrolyte sample through the sample input port 895 until itintersects roughly the chamber, and then by movement of thehigh-electrolyte medium through the alternative input port whilepreventing back-pressure from moving the low-electrolyte medium 882 backinto the sample input port 895. It should be noted that while theinterface 890 is shown as sharp and perpendicular to the sample inputport, the specific orientation and position of the interface 890 can bevaried within the present invention. Also, the differences in the rateof movement of bacteria between the high-electrolyte medium and thelow-electrolyte medium are related to the ratio of the conductivities inthe two media. It is preferable for the difference in conductivities tobe greater than 10-fold, and even more preferable for the difference tobe greater than 50-fold, and even more preferable for the differences tobe greater than 200-fold.

A cathode 910 is placed in the sample well within in the sample inputport 895, distal relative to the chamber from much or all of thebacteria 830 and 840. An anode 900 is placed after the last capturesurface 820. The placement of the anode 900 and the cathode 910 can bevaried within the operation of the present invention, but it should besuch that the bacteria 830 and 840 and capture surfaces 820 should bebetween the anode 900 and cathode 910. Indeed, both the anode 900 andcathode 910 can be outside of the chamber.

In the first step of operation, shown in FIG. 34B, a potential isapplied between the anode 900 and cathode 910. Because the resistance inthe high-electrolyte medium 880 is very low, the potential drop isprimarily through the low-electrolyte medium 882. Hence, the bacteria830 and 840 will move quickly through the low-electrolyte medium, untilthey reach the interface 890, at which point their movement issignificantly slowed. Indeed, by the use of a large difference in theconductance of the two electrolytes 880 and 882, it is possible for themovement in the two electrolytes to differ in movement by many orders ofmagnitude. Thus, the bacteria 830 and 840 will tend to concentrate atthe interface 890 as shown in FIG. 34B.

In FIG. 34C, the bacteria 830 and 840 are moved in a reverse direction(back towards the electrode 910) by reversing the potential, so as tomove the bacteria away from the interface. The distance that thebacteria can be moved can be quite short (e.g. hundreds of microns) orfar (e.g. centimeters) within the present invention. At this point, thehigh-electrolyte medium is removed by pushing low-electrolyte medium inthrough the alternative port 896, such that the entire system is nowlow-electrolyte medium, and the system is in a similar position to thatshown in FIG. 33A, with the bacteria to be introduced into the system.

Another embodiment of the present invention is shown in FIGS. 35A-D,which are side-view schematic diagrams of a chamber in whichcontaminating material is distinguished on the basis of its behaviorunder electrophoretic fields. In this case, four types of material areshown, including bacteria 830 and 840, as well as a first contaminant837 and a second contaminant 839. In FIG. 35A, all four materials aretransported to the loading surface 810, resulting in a situation similarto that of FIG. 33B. In this case, the loading surface 810 is set suchthat it has an attraction for the materials 830, 840, 837 and 839, andthat all of the materials bind to the surface 810.

In FIG. 35B, the polarity of the electrodes 815 and 816, such that thematerial is directed towards the electrode 815. The material 837 haseither a generally lower attraction to the loading surface 810, orexperiences a larger electrostatic force relative to the othermaterials, such that it is removed from the surface 810 while the othermaterials remain attached. In FIG. 35C, the potential on the electrodes815 and 816 is increased such that the bacteria 830 and 840 are removedfrom the loading surface 810, but the material 839, having a higherattraction for the surface 810, remains bound to the surface 810. Thebacteria 830 and 840 are now able to be transported through the chamber805, and to attach to the capture surfaces 820.

It should be noted that the binding force required to remove bacteriafrom a surface can be varied by careful selection of materialscomprising the loading surface 810 or the capture surface 820. Forexample, for nonspecific binding, the concentration of the nonspecificbinding agent (e.g. polyethyleneimine or other polycation) can bevaried, the length of the polymer chain can be varied, the type of ioniccharge can vary (e.g. primary, secondary, tertiary or quaternary amine,or the groups substituent to the nitrogen), the linear or volumetricdensity of the ionic charge in the polymer, or other changes. Inaddition, in the case of specific binding, if the binding agent (e.g. anantibody) does not provide in itself sufficient binding force to holdthe bacteria in place during the operation of the system, the specificbinding agent can be supplemented by a more tightly binding nonspecificagent, so that the total binding force is a combination of nonspecificand specific forces. It is also within the spirit of the presentinvention for there to be a sequence of capture surfaces 820 that aredistinguished not on the basis of different specific binding (e.g. byantibodies or aptamers), but rather by the different levels ofnonspecific binding, to which different bacteria bind with overalldifferent affinities. Thus, in general, the first capture surfaceencountered by the bacteria would have overall lower nonspecificbinding, and subsequently encountered surfaces 820 would then haveincreasing levels of nonspecific binding.

Organism Detection

Detection of the organisms can take place in a variety of differentmeans, though it is generally performed by visual detection means. Inthis case, a magnified image of the detection surfaces 820 are obtained,with or without the addition of stains, and this image is preferablyanalyzed by automated electronic means. For more general discussions ofdetection in the present invention, see also above.

The detection can include the use of methods of microscopy, includingbrightfield, darkfield, fluorescence, chemiluminescence, phase,differential interference contrast and other methods, as well as methodsof measuring overall light intensity and spectral response withoutimaging. Such methods can be further enhanced using illumination fromdirected laser or incoherent light illumination without the use ofconventional condenser illumination, such as can be used for scatteredlight or fluorescent light response. In addition, reflected light,transmitted light, or evanescent light illumination can be employed.While the methods of microscopy can be employed in the presentinvention, it is advantageous for the system to use optical systems thatdo not require careful and repeated calibration. Therefore, it ispreferred that optics employing a large depth of field are employed, andwhich are relatively low magnification. In addition, it is within theteachings of the present invention for multiple methods to be utilizedon the same sample, including, for example, the use of brightfield phaseimaging and fluorescence reflected imaging to be performed sequentially,in order to obtain different information about the same imaging field ofview.

The system frequently will involve the horizontal movement of either thechamber in which the bacteria are captured, or the detector, given thatthe measurements will be made over a significant period of time, andgenerally involving hours for those measurements involving bacterialgrowth (see below). In those cases, it is convenient for the system tobe able to reestablish its original relationship of bacteria relative tothe detector, so that images obtained over a period of time can becompared. While this can be sometimes performed with an “open loop”control system, in general, a “closed loop” system involving feedback ispreferred. Two preferred methods for this feedback involve the use ofvisual fiducials on the chamber, which fiducials are easily detected bythe visual system, with such information being used to adjust thehorizontal movement of the system until the original relationship of thechamber to the bacteria is established. A second method of convenienceis to make a rough “open loop” mechanical estimate of the originallocation, to obtain an image, and then at that time use image analysisto register the bacteria and other visual aspects of the chamber(potentially also involving visual fiducials). Such forms ofregistration can involve the use of Fourier transform or othercorrelation methods (such as matrix shifting) to match the images.

In general, the system will detect the presence and characteristics oforganisms through an automated program, such as the LabView softwarewith the IMAQ vision software from National Instruments (Austin, Tex.),ImagePro scripts, or high-speed image analysis using custom computersoftware. The system will store not only the presence of a bacterium,but also the location of the bacterium. Given that the bacterium isfixed at a location on a capture surface 820, it is considered that overa period of time, including growth of the organism, the bacterium willnot move significantly. Additionally, if a bacterium is noted in alocation at which a bacterium was not previously located, it is assumedeither that this bacterium was dislodged from another location, or thatthis bacterium was newly grown from another bacterium. Furthermore,changes in the size, the staining with moral or vital stains, or otherfactors can be correlated then to the change in status of the organismthat was previously seen in that same location.

In general, there will be at least one nonspecific capture surface 820,in order to capture all organisms that the specific capture surfaces 820do not capture. As mentioned before, this surface is preferably apoly-cationic surface, given that most bacteria have an overall negativeelectrostatic charge, or can be made to have such a charge at anappropriate pH. However, surfaces that have polyanionic charge,hydrophobic characteristics, single or multiple antibodies againstbacteria, glycoprotein binding agents, as well as polycationic and otheractive components, singly or in combination, can also be used. It isalso possible within the present invention for there to be only a singlecapture surface 820, and it is preferred in that case that the surfacehave nonspecific binding characteristics.

In the case of a nonspecific capture surface, it is still desirable tobe able to identify one or a number of different organisms. Thisidentification will generally be performed by indicators that arespecific for a serotype, genus, species, class, or other subset ofbacteria or other organism that is being detected, and is convenientlyan antibody, aptamer, or other molecule. The use of such indicators inthe present invention is demonstrated in FIGS. 36A-E, which areside-view schematic diagrams of detection of multiple bacteria on anonspecific surface. Note that the anode and cathode are not indicatedin this figure.

In FIG. 36A, bacteria 830 and 840 are bound to a nonspecific surface 825in the chamber 805. An indicator 842 that is specific for the bacteria840 is introduced into the chamber in the FIG. 36B through fluid flowthrough input port 802 to output port 803. This indicator 842 binds tothe bacteria 840, and then the unbound indicator 842 is removed from thechamber as in the FIG. 36C. It should be noted that the bonding of theindicator 842 to the bacteria 840 can be accelerated through use of theanode and cathode (not shown) that can be used to accelerate the bindingof the bacteria 830 and 840 to the surface 825. At this point, thepresence of the indicator 842 is determined through the detectionmethods as described above, and the specific locations are recorded inthe system (e.g. in a list, database, or other format that can either bestored in computer memory and/or on some physical storage medium such asa hard disk drive). In FIG. 36D, a second indicator 832 that is specificfor the bacteria 830 is introduced into the cell 805 and allowed to bindto the bacteria 830, and is then removed by fluid flow through the ports802 and 803, leaving the system in the state of FIG. 36E.

It should be noted that the means of detection means used to detect theindicator 832 and the indicator 842 can be the same. For example, if theindicators 832 and 842 are detected using fluorescence, the samefluorescent dye can be used in both detections. That is, at the state ofFIG. 36E, the system can detect the presence of both the indicators 842and 832 together. Because it has previously established the location ofthe bacteria 830 by determining the locations of the indicator 832 as inthe FIG. 36C, then the locations of the bacteria 840 will be in thosenew locations at which the indicators 832 and 842 are detected. Indeed,this method can be extended serially to allow the detection of a largenumber of specific bacteria using specific indicators, even though thebacteria are immobilized on a nonspecific surface 825.

In those cases where the means of detection are different (e.g. wherethe indicator 832 is detected by the fluorescence of one fluorophore,whereas the indicator 842 is detected by the fluorescence of anotherfluorophore, separable by excitation and/or emission wavelengths), thenthe indicators 832 and 842 can be introduced into the chamber 805simultaneously, washed out of the chamber simultaneously, and thendetected serially or concurrently.

It should be noted that in addition to the use of agents thatdistinguish specific bacteria (e.g. through the use offluorescent-labeled antibodies), there are many other characteristicsintrinsic to the bacteria or organisms that can distinguish them. Suchother characteristics include the morphology of individual bacteria(e.g. spherical versus rod versus helical), colony morphology (e.g. aclumped vs. chained), absorption or scattering of different lightfrequencies, their resistance or susceptibility to different classes ofdrugs (e.g. see below), their ability to grow in a particular growthmedium, their rate of growth, their size, and more. These agents can beused to distinguish multiple types of bacteria bound to a nonspecificcapture surface 820. Given also that there are frequently contaminantsin the sample that will give rise to signals with, for example, lightscattering or optical absorption means of detection, these methods canalso be used to distinguish bacteria from non-bacterial contaminants.

While the system can operate through the identification and monitoringof specific bacteria, it is also within the spirit of the presentinvention for the detector to sum the total response of all of thebacteria on the capture surface 820 (e.g. the scattered light). This canalso be used to indicate the total number of bacteria, and growth in thenumber of bacteria will be evidenced by an increase in the totalresponse.

Detection of Organism Viability

Organism viability can be determined by a variety of methods, and caninclude both methods that highlight viable organisms (vital stains) aswell as dead organisms (mortal stains). These stains can compriseethidium or propidium dyes, hexidium iodide, SYTO nucleic acid stains,7-aminiactinomycin D, SYTOX Green/Orange/Blue nucleic acid stains, andothers. A good introduction to these and other stains are available fromthe Molecular Probes Handbook, at www.probes.com.

It can be useful to detect the presence of new organisms or the increasein size of existing organisms. A method for accomplishing this is shownin FIGS. 37A-D, which are schematic diagrams of detecting growth in anorganism.

In FIG. 37A, bacteria 831 are attached to a nonspecific surface 825. Thebacteria 831 have a number of sites 843 for the binding of a molecule844. These sites 843 could represent regions of high negativeelectrostatic charge, glycoproteins, epitopes for broad or narrow rangeantibiotics, etc. In the FIG. 37B, the sites 843 are bound by themolecule 844 in a great excess of the molecule 844, so that all of thesites 843 are occupied by the molecule 844, after which the excessmolecule 844 is washed away.

In FIG. 37C, the bacteria 831 experience growth, either in size, or asindicated in the figure, in the number of bacteria 831, creating newbacteria 833. It should be appreciated that new bacteria will often bebound to the surface 825 close to the location of the original bacteria831, and that the proximity can be improved by the use of anelectrophoretic force during growth that drives the bacteria 831 and 833towards the surface 825. This proximity is not necessary to detect newbacterial growth 833, but rather to associate the new bacteria 833 withthe bacteria 831 from which they were derived, in order to demonstratethe viability and growth of the bacteria 831.

The new bacteria 833 will have binding sites 843 to which molecule 844is not bound. In the diagram, all of the sites 843 which are not boundby the molecule 844 are located on the new bacteria 833, while dependingon the manner of bacterial growth, it is also possible that thosebinding sites 843 will be distributed on both daughter bacteria arisingfrom the fission of the original bacterium 831. It should also be notedthat even in the absence of separation between new bacteria—for example,that the surface area of the bacteria 831 has increased, without thecreation of new bacteria 833—the increase in surface area will generallyinvolve the creation of new sites 843.

In FIG. 37D, the bacteria 831 and 833 are now incubated with themolecule 844 which is optionally modified so that it can be detectedwith an indicator 846 that can be detected by the system, and then themolecule 844 with indicator 846 that is not bound to the bacteria 831and 833 are washed away. Thus, any indicator 846 will be indicative ofnew bacterial growth or change in the number of sites on the bacteria towhich the indicator 846 can bind.

Organism Growth

The bacteria can now be grown in order to determine their viability,growth characteristics, and susceptibility to various agents (such asantibiotics). The growth occurs by the incubation of the bacteria in thepresence of a suitable medium at proper temperatures and oxygensaturation or depletion (e.g. for anaerobic or aerobic bacteria,depending generally on the source of the sample). The incubation mediumwill be in general matched to the bacteria being monitored—for example,lung aspirates, urine samples and blood samples would all be incubatedwith media that are well suited for bacteria or other organisms of therespective origins, as is well known in the art. In addition, theanti-growth agents to be tested for effects are also well-known in theart, and will change with the discovery of new agents and as the mix ofcurrent agents in use changes with the advent of resistance.

During the growth of the bacteria, it can be convenient to apply acontinuous or frequent electrophoretic force, in order that daughter ornew bacteria 833 are in roughly the same location as the originalbacteria 831 from which they are derived, allowing the provenance of thebacteria 833 to be determined. This will then allow the determination ofwhich of the original bacteria 831 are growing, and it secondarilyallows the determination of the type of bacteria to the new bacteria 833without having to do additional tests (e.g. antibody staining)

It should be noted that the electrophoretic force experienced by thebacteria is inversely related to the conductance of the medium, andtherefore it is convenient to have a low conductance growth medium. Mostmedia used for the growth of bacteria, yeast, and other organisms,however, generally has Na⁺, K⁺, Mg⁺², Cl⁻, SO₄ ⁻², NO₃ ⁻ and other ionsas both nutrients as well as to maintain an ionic strength of themedium. It is preferable for the growth medium to have a conductivity ofless than 5 mS/cm, and more preferable for the growth medium to have aconductivity of less than 2 mS/cm, and even more preferable for thegrowth medium to have a conductivity of less than 1 mS/cm. It should benoted that these conductivities are generally higher than that used inmovement of bacteria and other molecules for concentration of these atthe electrode, as described above. However, because the bacteria 833 arecreated at or proximal to the electrodes, the movement required is smallin distance and lower amounts of electrophoretic force are required. Inaddition, the application of the electrophoretic force need not beconstant, and can be applied intermittently, especially in those caseswhere the growth medium is not under constant bulk movement. Because ofthe slow diffusion of microorganisms, it is preferable to applyelectrophoretic force when the medium is not in bulk movement no morefrequently than every 10 seconds, and even more preferable no morefrequently than every 60 seconds. In general, many growth media containlarge amounts of salt (e.g. 0.5% NaCl in L Broth), and it is preferredthat this salt be replaced by a zwitterionic species, such as alanine orcysteine, that contributes very little conductance. It is alsopreferable for the osmotic strength of the medium be high enough so thatthe bacteria do not undergo osmotic shock. Non-ionic osmotic components,such as glycerol or sucrose, can be used for this purpose.

Growth by itself indicates primarily the viability of the organism, andpotentially the relative rates of growth of the bacteria. However, itcan also be used to study the susceptibility of the organism to variousanti-organism agents such as bactericidal and bacteriostatic agents.Examples of such agents include individual agents or combinations ofagents selected from antibiotic families such as cephalosporins,penicillins, carbapenems, monobactams, other novel beta-lactamantibiotics, beta-lactamase inhibitors, fluoroquinolones, macrolides,ketolides, glycopeptides, aminoglycosides, fluoroquinolones, rifampin,and other families, including novel agents, used as antibiotics inclinical practice or in research. In the simplest case, this wouldinvolve the incubation of the organism in a constant concentration ofanti-organism agent (AOA), and determining the rate of growth and/or therate of death of the organism.

FIG. 33E shows how this would be performed with the present invention.In FIG. 33D, the bacteria 830, 835 and 840 have been specifically boundto capture surfaces 820. After a period of incubation in oneconcentration of an AOA in a growth medium (indicated by lightstippling), the bacteria 840 have increased in number, and the bacteria830 and 835 have not, indicating that the bacteria 840 are notsusceptible to AOA at the concentration used, and that the bacteria 830and 835 are susceptible at the concentrations of the AOA used. It shouldbe noted that bacteria 835 are of the same type as bacteria 830, exceptthat they are dead. Given a mortal or vital stain, therefore, it can bedetermined that bacteria 830 have not been killed by the concentrationof AOA, indicating either that AOA prevents growth but does not kill thebacteria 830, or that at the concentrations used, AOA only acts to stopgrowth.

In FIG. 33F, the concentration of the AOA is increased, and the numberof bacteria 840 still increases, indicating that the bacteria 840 arenon-susceptible to the bacteria even at this concentration. However, nowthe bacteria 830 have been killed (indicated by the dead bacteria 835),indicating that at this concentration, AOA is lethal. Thus, as indicatedin FIGS. 33E-F, by using increasing concentrations of the AOA in thegrowth medium, the concentration response of the bacteria to the AOA canbe determined. Clearly, by increasing the amount of AOA in steps over aperiod of time, the minimum inhibitory concentration (MIC) can bedetermined. In addition, because viability of the bacteria can also bedetermined at each concentration, the minimum bactericidal concentration(MBC) can also be determined.

It should be noted that the detection of growth and viability atdifferent concentrations of AOA can be performed either by using aseries of chambers 805 in the cell 804, each of which challenges thebacteria with a specific concentration of AOA, or alternatively, byincreasing the concentration within a given chamber 805. In the formercase, the time response of the bacteria can be easily established, aswell as the persistent response of the bacteria once the AOA has beenremoved (e.g. a post-antibiotic effect). That is, the bacteria can alsobe challenged with a given concentration of AOA for a brief period, andthen the medium replaced with a medium lacking the AOA, and the lack ofgrowth or the death of the bacteria can be monitored over time.

As described above, so as not to use separate chambers 805 for everydifferent concentration of AOA, the concentration of AOA within achamber 805 can be increased over time. FIGS. 38A-B are graphs of theresponse of bacteria to changing concentrations of AOA. In FIG. 38A, theconcentration of AOA is increased over time, generally according to anexponential increase with time, although it is also convenient for theconcentration to increase linearly or according to otherconcentration/time relationships, including step functions increasingthe concentration; these step functions can be placed at regularconcentration intervals, or alternatively at standard concentrations asindicated or suggested by clinical laboratory standards as might be setby organizations such as the National Committee for Clinical LaboratoryStandards. The system is then used to determine the total number ofbacteria, the number of dead bacteria, and the number of live bacteria(as described above, any two of these numbers gives rise to the thirdnumber).

At the point that the total number of bacteria does not continuegrowing, indicated in the figure at the concentration A, is consideredto be the MIC. The point where the number of live bacteria begins todecline (at the concentration B) is considered to be the MBC. It shouldbe noted that the actual MIC and MBC can be lower than theconcentrations A and B respectively, and will only be the MIC and MBC inthose cases where the rate of increase in concentration is very slowrelative to the growth of the bacteria. Thus, given that it is desiredthat the MIC and MBC of AOA be determined within a factor of X, it ispreferable for the concentration of AOA to increase by a factor of X nofaster than half the doubling time of the bacteria under the conditionsof the incubation lacking AOA, and it is more preferable for theconcentration of AOA to increase by a factor of X no faster than thedoubling time of the bacteria, and it is most preferable for theconcentration of AOA to increase by a factor of X no faster than twicethe doubling time of the bacteria.

The less growth of bacteria required in order for there to be highconfidence that growth has occurred will reduce the time needed toperform a test. By monitoring individual bacteria, growth can be seenwith the doubling of only a small number of bacteria. That is, if lookedat in bulk as in conventional turbidity assays, for example, the limitof sensitivity of detecting bacterial growth is limited by the signal tonoise ratio in the turbidity measurement. However, the fission of abacterium is a discrete event that can be detected, even if thatbacterium is one of many thousands of bacteria. Thus, the presentinvention can have a very high sensitivity, with the system preferablyable to detect doubling of less than 25% of the bacteria, morepreferably able to detect doubling of 10% of the bacteria, and mostpreferably able to detect doubling of 5% of bacteria. Note that thedoubling time for a fraction of the bacteria can be either predetermined(e.g. by calibration in a laboratory with experimental specimens), ormore preferably, by comparing the bacteria in the absence of the AOAwith those in the presence of the AOA—this makes the results internallycontrolled.

The measurement cut-off points for determining antibiotic susceptibilitycan, as discussed above, be expressed in absolute terms, such as thedoubling of a given percentage of the bacteria. However, the number ofbacteria required to make a statistically valid judgment can bedependent on the number of bacteria present in the sample. For example,if there are only 10 bacteria present in each chamber, evidencing asingle bacterium doubling represents 10% of the sample. Alternatively,with very large numbers of bacteria on the surface (e.g. more than100,000), the doubling of even 1,000 bacteria (i.e. 1%) is probablystatistically significant. Thus, it is in many cases preferable toanalyze the number of bacteria required to show doubling in the controlcondition (i.e. growth medium absent the AOA) relative to the number ofbacteria showing doubling in the experimental condition (i.e. growthmedium with the AOA) as to be statistically relevant. For example, aconventional method would be to apply a chi-squared test to these twonumbers, and to decide whether the results met a particular probabilityof significance. In general, it is preferable for this probability to beless than 0.05, and even more preferable for this probability to be lessthan 0.025 and most preferable for this probability to be less than0.01. Because small numbers of bacteria will not permit very smallchi-squared probabilities, the standards for probability can beconveniently reduced for cases of very small numbers of bacteria (e.g.less than 20 viable bacteria in the growth medium control).

It should be understood that the doubling time of bacteria is apopulation phenomenon, and that within a population of bacteria, somebacteria will divide more quickly than others. This could be due both toslight genetic differences in a population, or purely statisticaleffects. However, it can also be due to the stage at which eachbacterium is growing during its preparation, as the bacteria willexhibit substantially different lag times in their growth when placed innew medium depending on that stage. While a longer period of time isgenerally going to provide more information about the growthcharacteristics and AOA susceptibility of the bacteria, there is a needto supply to medical personnel information about the bacteria and theirsusceptibility to AOAs. Given that lag time for most of the bacteria ofinterest is on the order of 2-6 hours, and the doubling time of thebacteria is generally 1-2 hours, it is preferable for measurements ofbacterial growth and susceptibility to AOA use detection of the bacteriaat no more than 8 hours, and more preferably less than 6 hours. Even ifnot all bacteria in a sample have an opportunity to demonstratedoubling, a large enough fraction of those bacteria will have so as tobe able to indicate susceptibility and growth.

In this case, it is useful to have all information available forindividual bacteria relating to vital and/or mortal staining (indicatinglive versus dead bacteria), as well as growth in the presence of growthmedium with and without the presence of AOA. Any observation in whichthe fraction of live bacteria decreases by a first predeterminedfraction in the presence of AOAs, or in which the growth of bacteria(evidenced either by doublings or by increases in the size of thebacteria) is decreased by a second predetermined fraction in thepresence of AOA, are evidence of the action of the AOA. In general, thefirst determined fraction, because of its evidence of higher death, willgenerally be smaller than the second predetermined fraction. Apreferable value of the first predetermined fraction is 20%, and a morepreferable value is 33% and the most preferable value is 50%. Apreferable value for the second predetermined fraction is 50%, and amore preferred value is 66%, and a most preferred value is 80%.

As indicated above, most studies on AOA susceptibility relate to theconcentration at which a particular effect is encountered, rather thanthe specific kinetics and effects that are observed. That is, inconventional tests, the bacteria are usually challenged with a number ofdifferent concentrations (or even changing concentrations) of AOAs todetermine the concentration at which the bacteria exhibit death orlowered rates of growth, from which the MIC or MBC can be determined.Consider, for example, a conventional antibiotic test employing an agarplate with an antibiotic disk. Around the disk are colonies of varioussizes, representing not simply death, but slower growth in the presenceof differing concentrations of antibiotic. By this measure, the MIC isnot easy to define, since incubating the plates for an extended periodof time would allow colonies to appear at concentrations that areconsidered inhibitory.

However, both from a standpoint of time and cost, it can be convenientin some cases to instead challenge the bacteria with single, constantdoses of the AOA, and then to observe the specific effect and rate ofeffect of the drug, in order to determine susceptibility. In the presentinvention, a constant dose of AOA can be provided, and the rate at whichbacteria are killed, or the degree to which their growth is reduced, canbe used to gauge the likely effects at a multiplicity of therapeuticdoses. These responses can be described with new measures of AOA effect,such as the bacterial doubling time in the presence of an AOA divided bythe bacterial doubling time in the absence of the AOA. In this case, forbacteria that are resistant to an AOA but whose doubling time is tripledin the presence of the AOA, treatment with the AOA can still bemeaningful. These values can be provided either at a single dose, or atmultiple doses. To the extent that bacteria of differing levels ofsusceptibility can be isolated and studied, the information at one ormore concentrations of the AOA can be useful in then predicting theresponse at other concentrations.

It should be noted that the concentration of AOA in a human or animal isdetermined by the amount and frequency of treatment (e.g. injection), aswell as the AOA pharmacokinetics. In many cases, the pharmacokineticsare well-known for disease-free humans, and can be modeled on the basisof the known medical state (e.g. liver failure) of the person beingmonitored. Using this information, the concentration of AOA over time inthe target organ (e.g. blood, urinary tract, lungs) can be estimated.This AOA concentration can be approximated in the chamber by mixingmedium with AOA in relative parts with medium lacking AOA, to producethe estimated profile of AOA such as that shown in FIG. 38B. In general,the concentration of AOA will rise, peak, and then exponentially decay.As before, the total number of bacteria, the dead bacteria and the livebacteria can be monitored over time. In this case, the pharmacodynamicparameters MIC and MBC are not well defined, since one is looking at theresponse to the bacteria including the pharmacokinetics of AOA, and onelooks therefore at the minimum inhibitory dose and the minimumbactericidal dose by running replicates of the system at differentdoses, and then monitor if the overall AOA concentration profile resultsin the cessation of growth or the death of the bacteria. It should benoted that while the analysis of FIG. 38B deals with only a single doseof AOA (i.e. rise, peak, decay), it is also possible to continue theanalysis on sequential doses of AOA as would often be used in treatment(e.g. injection 4 times daily).

It should be noted that the methods of the present invention can beapplied not only to the response of organisms to AOA, but also theresponse to other conditions, such as hormones, drugs (e.g. for drugsensitivity testing), environmental or other agents. These agents can beso analyzed, as long as the response is detectable by the detectoremployed. In many cases, a stain of some sort may be required in orderto make the response to the condition visible.

In the discussion above, the timing of the application of AOA can berelated either to the time at which the bacteria are first placed intogrowth medium, or alternatively, to the time at which bacterial growthis first detected (e.g. through changes in the size of the bacteria, orthe presence of daughter cells). In the latter case, growth can bemonitored continuously, and AOA added to the incubation at such time asit is determined that the lag time has completed. The completion of lagtime will generally be that point at which some predetermined fractionof cells have shown signs of growth, which is preferably less than 50%of cells, and more preferably less than 30% of cells, and mostpreferably at less than 20% of cells.

Examples of the use of microscopy to demonstrate cell growth areprovided by J. R. Lawrence, D. R. Korber, and D. E. Caldwell (1989)“Computer-Enhanced Darkfield Microscopy for the Quantitative Analysis ofBacterial Growth and Behavior on Surfaces”, J. Microbiol. Methods 10:123-138 and A. Elfwing, Y. LeMarc, J. Baryani, and A. Ballagi (2004)“Observing Growth and Division of Large Numbers of Individual Bacteriaby Image Analysis”, Applied and Environmental Microbiology70(2):675-678. It should be noted from Elfwing et al. that growth ofbacteria can be measured under laminar flow whereby daughter cells aresheared away, giving a sawtooth optical profile in which the cell sizeincreases, and then with the removal of the daughter cell, the cell sizeabruptly declines. In the present invention, in addition to cell size(e.g. the number of pixels), the amount of fluorescence or the amount oflight scatter can also be used.

Transport to the Detection Surface Using Alternative Means

Above, the transport to the detection surface using electrophoreticmeans has been discussed; other means are discussed here. For example,the bacteria can be transferred to the detection surface usingcentrifugal force. FIG. 39A is a schematic view of a centrifuge tube 900modified for the concentration of bacteria onto a capture surface 905,and FIG. 39B is a cross-sectional view of the centrifuge tube 900 ofFIG. 39A. The tube 900 comprises three separable pieces, a sample tube903, a capture piece 910, and a bottom piece 912. The sample tube 903comprises an outer structure 904 that is a cylinder of diameter thatfits snugly into a centrifuge fixture for a centrifuge capable ofdelivering centrifugal force preferably above 200×g, and more preferablyabove 1000×g and even more preferably above 2500×g. The sample tube 903further comprises an inner structure 907 that contacts the outerstructure 904 for purposes of strength, and the inner structure 907 haseither a square or a rectangular cross section. It should be noted thatthe sample tube 903 will hold a sample 916 containing a bacterialsample, which when centrifuged will deposit the bacteria in the sampleonto a capture surface 905 that is preferably either square orrectangular (although other shapes are allowed in the presentinvention), and whose shape matches the shape of the inner structure907. The cross-sectional shape of the inner structure 907 is limited bythe shape of the capture surface 905, and instead of having an innerstructure 907 and an outer structure 904, there can be only an innerstructure 907 given either that the centrifugal force and sample tube903 materials are such that the inner structure 907 can maintain itsdimensional integrity without need for the outer structure 904, or thatthe centrifuge fixture into which the centrifuge tube 900 fits isroughly matched to the shape of the tube 900 (e.g. is square orrectangular).

The sample tube 903 fits snugly onto the capture piece 910, which caninclude a gasket 914 so that under centrifugation, the bacterial sample916 is not forced from the sample tube 903. The sample tube 903generally has interfaces for both the sample tube 903 and the bottompiece 912, and has a top surface in contact with the bacterial samplewithin the sample tube 903 that has a capture surface that generally hasnonspecific binding for bacteria or other organisms on the sample. Suchnonspecific surfaces have been described in detail above. It should benoted that the capture surface can either be placed directly onto anintegrated capture piece 910 (for example, a molded plastic piece), oralternatively can be a removable top piece that, on removal, is a flatsquare or rectangular piece that is preferably between 100 microns and1500 microns in thickness and is made of a suitable plastic or glass.The following discussion relates to an integrated capture piece 910.

The bottom piece 912 is molded to fit snugly into the centrifuge fixturefor the centrifuge used, and is typically hemispherical or conical. Onceagain, depending on the centrifuge fixture, the shape of the bottompiece can be various. Furthermore, if the centrifuge fixture is flat onthe bottom, the bottom piece 912 can be dispensed with, and the bottomsurface of the capture piece 910 would contact then the bottom of thecentrifuge fixture.

Upon the bacteria in the sample 916 being centrifuged onto the capturesurface 905, the capture piece 910 is separated from the sample tube903, and placed between a top fixture 922 and a bottom fixture 924 asshown in FIG. 39C, a cross-sectional side-view of a detector 930 usingthe capture piece 910 of FIGS. 39A-B. The fixtures 922 and 924 are heldtogether with screws 932 and nuts 936 or other means (e.g. clamps), witha gasket 934 providing a water tight seal between the top fixture 922and the capture piece 910. In the top fixture 922, above the capturepiece 910, are a series of linear walls 923 that fit snugly onto thecapture surface 905 such that isolated chambers 920 are created abovethe capture surface 905. These chambers 920 have input and output ports(not shown) that allow the introduction of growth media, AOA, indicators(e.g. fluorescent antibodies) for cell type, mortal and vital stains andother such media as needed to execute the steps 730, 740, and 750 ofFIG. 6. Detection is provided through the top fixture 922; however, ifthe capture surface 905 is removable from the capture piece 910 asdescribed above, detection can take place through the capture surfacegiven a suitable fixture.

An alternative embodiment is provided in FIGS. 40A-B, which are across-sectional top-view and side-view of a filter-based detectiondevice 950 that uses a porous capture filter 960, with the FIG. 40Bbeing shown through cross-section W of the FIG. 40A. The device 950comprises a series of detector channels (of which four are shown, butwhich can comprise tens of channels), each of which comprise the porousfilter 960, which in conjunction with a separator 962, separates anupper chamber 964 and a lower chamber 966. The filter 960 is at the endof the channel, with movement of medium in through an input port 952,across the upper surface of the separator 962, through the filter 960,back across the lower surface of the separator 962 and then out throughan output port 954. The channel is bounded on other sides by outerstructure 970, which can comprise a single piece (as shown), or a toppiece bonded onto a bottom piece, wherein the top piece, lying above thefilter 960, is generally transparent so that bacteria bound to the topsurface of the filter 960 can be detected visually. While there can be aseparate output port 954 for each channel, the output ports can beshared, as is shown in the figure.

The filter 960 can comprise a track-etched membrane (e.g. polycarbonate,polyethylene terephthalate, glass, aluminum or other material), aluminumoxide, Teflon®, nitrocellulose, and other materials. In certain cases,the filters 960 are manufactured separately, being of different materialfrom that of the separator and the outer structure 970, and aretherefore placed onto the porous structural element (not shown) thatholds the filter into place and prevents media from flowing from theupper chamber 964 to the lower chamber 962 without going through thefilter 960.

The filter preferably has median pore size less than 500 nm, and morepreferably less than 250 nm and most preferably less than 100 nm, whichwill generally be smaller than the smallest bacterial organism to bedetected. In general, such pores are difficult to make substantiallyless than 50 nm, and for very small organisms (e.g. virus particles)that are on the order of or smaller than the diameter of the pores, itis convenient to bind particles to the organisms (e.g. particlescomprised of polystyrene or other polymer, gold, ceramic, or othermaterial) using antibodies, aptamers, or electrostatic attraction (e.g.where the particles are covered with a polycationic surface), such thatthe combination of organism and particles are larger than the pores. Itshould be noted, however, that these particles must not be used in suchlarge quantity such that when in packed configuration have an arealarger than that of the filter 960.

Bacterial samples are generally prepared as described above so as toremove particle contaminants (e.g. dust), mammalian cells, mucus, andother interfering agents, and in general to reduce the sample volume toa milliliter or less (the sample flow rates through the filter can bevery low in certain cases, such as track-etched filters). Bacteriaintroduced in a sample through the input port 952 flow across the filter960, and are captured on its surface. The bacteria are detected on thesurface through the outer structure 970 through means described above.Then, media comprising nutrients, mortal and vital stains, indicators,AOA, and other materials as described in sections on growth anddetection above are introduced through the input port 952 and removedthrough the output port 954. If it is desired that a constant force beplaced on the system such that bacteria that newly arise through growthin media do not move far from their place of origin, movement of mediumthrough the system can be maintained.

In another embodiment, sample concentration, transportation andattachment is achieved by simultaneously on a nonspecific capturesurface where multiple forces are applied to effect separation of thebacteria into differing fractions on the basis of size (volume orcross-section), charge-to-mass ratio, relative attachment ofelectrostatic or magnetic tags, electrophoretic mobility, and othercharacteristics. In one embodiment, the bacteria are moved horizontallyalong the chamber through movement of the fluid, which movement may beaccomplished via electroosmosis, positive displacement pumps,peristaltic pumps, or other means, or alternatively, the bacteria canmove under the application of a directional force (e.g. electrophoresis,magnetic fields, etc.). The vertical directional force on the bacteriamay be accomplish via fluid flow (e.g. via filtration), electrophoresis,electroosmosis, centrifugal or by other means. It should be noted thatthe force in any one direction can be the result of additive or opposingforces from one (e.g. fluid flow can be applied in opposite directionsat different cross-sectional locations), two or more of the forcesdescribed above. Also, the forces can be oriented so that they areparallel, orthogonal, or a combination of the two.

FIGS. 41A-B are schematic cross-sections of a detection system usingmultiple forces to effect separation of the bacterial sample. In FIG.41A, a combined horizontal and vertical fluid movement caused bypositive displacement pressure in inlet 803 produces flow out the exitport 802 in the chamber 805 via a track-etched filter 1001. This bulkfluid movement is coordinated with the further application of horizontalelectrophoresis using an anode 815 and a cathode 816, which opposes thefluid flow in a simultaneous or sequential manner. That is, insequential coordination, fluid movement can be performed for a certainperiod, and then followed by a period of fluid non-movement during whichelectrophoresis is applied, or the electrophoresis can be applied duringmovement in simultaneous coordination. In the latter case, the speed ofmovement or the magnitude of the electrophoretic force can be varied,such that bacteria of two types (denoted by stars 830 and 835 anddiamonds 840), clusters of the bacteria types (denoted by 830B and835B), and sample contaminants 1000 are separated by various physicalcharacteristics such as size, shape, and electrophoretic mobility.

In FIG. 41B, at the conclusion of the separation, the electrophoreticand bulk flow fluid forces have been balanced so that bacteria 830, 835and 840 and contaminants 1000 are separated on the basis of size andcharge. The bacteria 830 separate into regions of individual bacteria830 and clumped bacteria 830B. Also, there is a separation of livebacteria 830 from dead bacteria 835, which separation occurs due tochanges in size, surface properties, and charge (due in part to changesin permeability). These separate areas aid in the identification ofbacteria on the filter 1001 and the separation or removal ofcontaminants 1000 from the sample.

While the cathode 816 is in the upper part of the system shown (i.e. thecathode 816 and the anode 815 are on the same side of the filter 1001),it is also within the spirit of the present invention for the cathode816 to be placed into the lower part of the system shown, so that thecathode 816 and the anode 815 are on opposite sides of the filter 1001.In this case, bacteria moving across the filter 1001 are affected by afluid flow, which both moves the bacteria across the filter 1001 andeventually down onto the filter 1001, as well as an electrophoreticforce that moves the bacteria only downwards (and to the pores, throughwhich the electrophoretic force is applied). Thus, the forces of fluidflow and electrophoresis can be independently applied, effecting aseparation of bacteria and contaminants depending on their responses tothese two forces. Indeed, in this case, it can be convenient for theoutput port 802 to be in the upper part of the system, so that fluidflow forces are almost entirely horizontal, whereas the electrophoreticforce is vertical. The use of any permeable membrane supportingelectrophoresis can be used in this apparatus instead of the track-etchfilter 1001.

It should be noted that there are many configurations of the channels inthe device within the spirit of the present invention. For example,while there can be separate filters 960 for each channel, it isconvenient for there to be a single filter 960 which is separated bywalls between each channel. Furthermore, while the filter 960 is shownto be rectangular within each channel, it is also convenient for thefilter 960 to be in an aspect ratio (square or slightly rectangular)that matches the field of view of the optic system used to detectbacteria on the surface of the filter 960. Furthermore, while the inputports 952 and output ports 954 are shown on the same side of the device950, they can also be located on opposite sides of the device 950, ororiented perpendicularly to one another.

Many Embodiments within the Spirit of the Present Invention

It should be apparent to one skilled in the art that the above-mentionedembodiments are merely illustrations of a few of the many possiblespecific embodiments of the present invention. It should also beappreciated that the methods of the present invention provide a nearlyuncountable number of arrangements of indicators, tags, detectors,mixing means, force application means and more.

Some of the embodiments are described combinatorially in FIG. 42, ablock diagram of a biodetection by the present invention.

As shown in Target Identity, the targets can comprise DNA, RNA, protein,starch, lipids (e.g. steroid hormones), and may further comprisecombinations of these (e.g. glycoproteins). Furthermore, the targets cancomprise organisms or tissues, including bacteria, fungi, viruses,mycoplasma, protozoans, or various types of animal or plant cells (e.g.circulating cells, or tissue culture cells). More generally, the targetscan comprise any material or molecule for which a specific probe can bedeveloped.

In an optional Sample Preparation, the target in which the target ispresent can be prepared for subsequent analysis, for reasons that caninclude removal of contaminants, concentration to a more easily handledvolume, or placement of the targets into a buffer whose characteristicsare more appropriate for subsequent analysis steps. This samplepreparation can comprise centrifugation (either to centrifuge down thetarget from the sample for resuspension in another buffer or tocentrifuge out particulate contaminants from the targets in solution),ion exchange (e.g. filtration through an ion exchange resin or mixingthe sample with ion exchange beads), filtration, electrophoresis (e.g.stacking electrophoresis or gel electrophoresis with extraction), andother forms of biochemical, chemical or physical separation (affinitycolumns, phase partitioning, precipitation, etc.).

In an optional Tagging, the target is tagged so as to improve either itsmovement towards the probe, or to make it more detectable by thedetector. Those tags affecting mobility comprise electrostatic tags(e.g. for movement under electrophoretic fields), magnetic tags (e.g.for movement in magnetic fields), electrostatically polarizable tags(e.g. for movement in dielectrophoretic fields) and other tags withphysical properties that change the movement of targets in differentphysical or chemical environments. The tags can also comprise indicatortags, such as light scattering particles, electrochemical tags,fluorescent tags, upconverting phosphor tags, quantum dot tags, orenzyme tags (e.g. peroxidase) that will improve the visibility of thetagged target at a subsequent stage. It should be noted that the tag canincorporate both functionalities (movement and detection), either withina single entity (e.g. a light scattering particle that is alsoelectrostatically polarizable or a magnetic particle that scatterslight), or resulting from the bonding together of two different entitieswith different functionality.

It should also be noted that Tagging may occur multiple times, forexample in a first instance to enhance mobility and in a second instanceto enhance detection. Indeed, Tagging can occur either before the TargetCapture (discussed below), after Target Capture, or both before andafter Target Capture.

In Target Capture, the target is captured on a surface. This capturegenerally involves a movement of the target to the surface, and cancomprise electrophoresis, dielectrophoresis, centrifugation, magneticfield capture, filtration, gravity, or other forces that result in thecapture of the target on the surface. The surface can have either anatural affinity for the target, or can be treated in such a way as tohave a specific affinity for some targets (e.g. coating the surface withan antibody), or a general affinity for many targets (e.g. coating thesurface with a polycationic polymer).

In concert with the Target Capture, optional Horizontal Movement can beperformed, wherein the Horizontal Movement uses electrophoresis,filtration, bulk flow (e.g. from pumps or electroendoosmosis), or othermeans to affect the distribution of the target on the surface. Thedistribution can either be made more uniform (e.g. to allow the targetto come into proximity with more of the surface), or alternatively, canbe used to place targets with different characteristics at differentlocations on the surface (e.g. to “fractionate” bacteria on the basis oftheir electrophoretic mobilities).

In Washing, the unbound target is removed, and an attempt can be made toremove nonspecifically bound material. Washing can compriseelectrophoresis, dielectrophoresis, chemical (e.g. salt, pH, surfactant,affinity competitor), physical (e.g. temperature), magnetic field, orother means of affecting the binding of the probe (or nonspecificcapture agent) to the captured target. It should be noted that somefraction of the nonspecifically bound material can be more tightly boundthan that of the specifically-bound target. The washing can alsodistinguish specifically-bound target as that material that is releasedbetween two levels of stringency.

In optional Staining, the bound target can be stained in order to affectits visibility, and can be used to ascertain the state of the target.This is particularly useful in the case of cells (bacterial, animal orplant), where the use of mortal and vital stains indicate whether thecells are alive or dead, and the use of serotyping (generally with theuse of labeled monoclonal or polyclonal antibodies) can establish theidentity of the cells (e.g. genus, species, cell type). It should benoted that Staining can alternatively be performed as part of Tagging(e.g. a fluorescent tag can be attached via a serotype specificantibody), prior to Target Capture, between Target Capture and Washingor after Washing. The time at which Staining is best performed dependson the persistence of the stain, the degree to which the staininterferes with other steps, and other reasons.

Alternatively or in conjunction with the Staining is optionalIncubation, in which the target is incubated, which is generallyperformed with live targets (e.g. bacteria). In this case, theincubation is performed conveniently in a growth medium conducive togrowth, and can be accompanied with a biological condition, such as theapplication of an AOA, challenge with a hormone, drug, temperature, orother biological mediator. It is best if the expected response of thetarget to the condition is visible by the detector, which can involvethe use of a stain. Staining may also be employed after the Incubation,and it should be appreciated that the application of the stain can occurmultiple times in an analysis (e.g. so that cells that are newly grownin the Incubation can be stained with the stain, or so that the responseof the cells to the condition).

In Detection, the targets are detected by the detector. The detector canbe an optical detector, which can be an imager/camera, which can viewthe targets via brightfield, darkfield, frequency change (e.g.fluorescence, upconverting phosphors, quantum bits), phase, emittedlight (e.g. chemiluminescence), or other imaging means. The detectioncan also comprise a photomultiplier tube, in conjunction with a laserscanner, or with averaging optics that spread the light from the entirefield or a substantial portion of the field onto the light gather source(which could also utilize a photodiode, photoresistor or other lightmeasurement device). Also, the detection can involve SPR, either in animaging mode, or in averaging mode. It should be noted that there arenon-optical means of detection, using for example measurement ofelectrical current, which can be used with certain embodiments of thepresent invention.

In some instances, it is convenient to perform the detection multipletimes at different washing stringencies, in which case detection can befollowed by another cycle of washing and detection. Also, as shown, itcan be convenient to perform detection multiple times after continuedIncubation or multiple Staining (e.g. to determine the susceptibility oforganisms to AOA).

In Analysis, the data from the detector is analyzed. The Analysis cancomprise tracking individual targets, or measurement and analysis ofbulk properties of the signal generated by the detector. Additionally,the analysis can look at the change of signal over time (e.g. inresponse to the growth of organisms, their viability in differing AOAconcentrations, or target binding at different washing stringencies).

It should be noted that the embodiments of the present invention are notcomprehensively enumerated in FIG. 42, and that the multitudinousembodiments embedded combinatorially in the figure are illustrativeonly.

Numerous and varied other arrangements can be readily devised by thoseskilled in the art without departing from the spirit and scope of theinvention. Moreover, all statements herein reciting principles, aspectsand embodiments of the present invention, as well as specific examplesthereof, are intended to encompass both structural and functionalequivalents thereof. Additionally, it is intended that such equivalentsinclude both currently known equivalents as well as equivalentsdeveloped in the future, i.e. any elements developed that perform thesame function, regardless of structure.

In the specification hereof, any element expressed as a means forperforming a specified function is intended to encompass any way ofperforming that function. The invention as defined by such specificationresides in the fact that the functionalities provided by the variousrecited means are combined and brought together in the manner which thespecification calls for. Applicant thus regards any means which canprovide those functionalities as equivalent as those shown herein.

What is claimed is:
 1. A system for the identification of individualmicroorganisms in a sample comprising: an enclosed chamber comprising: afirst electrode disposed on a detection surface, wherein the firstelectrode is a transparent conductive surface; a second electrodedisposed on a second surface; an input port configured to transport afluid into the chamber; an output port configured to transport the fluidout of the chamber; a capture surface disposed on the first electrode,wherein the capture surface comprises a binding agent configured to bindthe individual microorganisms in spatially discrete locations; anelectrical controller operably linked to the first and second electrodesand configured to control potential between the first and secondelectrodes; an optical detector configured to detect the individualmicroorganisms bound to the capture surface; and a storage controllerconfigured to perform analysis of an image obtained by the opticaldetector.
 2. The system of claim 1, wherein the storage controllercomprises a computer configured with software suitable for performingimage analysis.
 3. The system of claim 1, wherein the storage controlleris configured to register the locations of individual microorganismsbound to the capture surface.
 4. The system of claim 1, wherein thestorage controller is configured to determine the morphology ofindividual microorganisms bound to the capture surface.
 5. The system ofclaim 1, further comprising a first indicator configured to bind to afirst microorganism.
 6. The system of claim 5, further comprising asecond indicator configured to bind to a second microorganism.
 7. Thesystem of claim 6, wherein the first indicator and the second indicatorare fluorophores distinguishable by the detector by excitationwavelength, emission wavelength, or both excitation and emissionwavelengths.
 8. The system of claim 2, wherein the system is configuredto perform detection and image analysis automatically.
 9. The system ofclaim 2, wherein the first electrode comprises an indium tin oxideelectrode.